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Sample records for 5-ht3a receptor knockout

  1. Agonist- and antagonist-induced up-regulation of surface 5-HT3A receptors

    PubMed Central

    Morton, Russell A; Baptista-Hon, Daniel T; Hales, Tim G; Lovinger, David M

    2015-01-01

    Background and Purpose The 5-HT3 receptor is a member of the pentameric ligand-gated ion channel family and is pharmacologically targeted to treat irritable bowel syndrome and nausea/emesis. Furthermore, many antidepressants elevate extracellular concentrations of 5-HT. This study investigates the functional consequences of exposure of recombinant 5-HT3A receptors to agonists and antagonists. Experimental Approach We used HEK cells stably expressing recombinant 5-HT3A receptors and the ND7/23 (mouse neuroblastoma/dorsal root ganglion hybrid) cell line, which expresses endogenous 5-HT3 receptors. Surface expression of recombinant 5-HT3A receptors, modified to contain the bungarotoxin (BTX) binding sequence, was quantified using fluorescence microscopy to image BTX-conjugated fluorophores. Whole cell voltage-clamp electrophysiology was used to measure the density of current mediated by 5-HT3A receptors. Key Results 5-HT3A receptors were up-regulated by the prolonged presence of agonists (5-HT and m-chlorophenylbiguanide) and antagonists (MDL-72222 and morphine). The up-regulation of 5-HT3A receptors by 5-HT and MDL-72222 was time- and concentration-dependent but was independent of newly translated receptors. The phenomenon was observed for recombinant rodent and human 5-HT3A receptors and for endogenous 5-HT3 receptors in neuronal ND7/23 cells. Conclusions and Implications Up-regulation of 5-HT3A receptors, following exposure to either agonists or antagonists suggests that this phenomenon may occur in response to different therapeutic agents. Medications that elevate 5-HT levels, such as the antidepressant inhibitors of 5-HT reuptake and antiemetic inhibitors of 5-HT3 receptor function, may both raise receptor expression. However, this will require further investigation in vivo. PMID:25989383

  2. Pathways and Barriers for Ion Translocation through the 5-HT3A Receptor Channel

    PubMed Central

    Di Maio, Danilo; Chandramouli, Balasubramanian; Brancato, Giuseppe

    2015-01-01

    Pentameric ligand gated ion channels (pLGICs) are ionotropic receptors that mediate fast intercellular communications at synaptic level and include either cation selective (e.g., nAChR and 5-HT3) or anion selective (e.g., GlyR, GABAA and GluCl) membrane channels. Among others, 5-HT3 is one of the most studied members, since its first cloning back in 1991, and a large number of studies have successfully pinpointed protein residues critical for its activation and channel gating. In addition, 5-HT3 is also the target of a few pharmacological treatments due to the demonstrated benefits of its modulation in clinical trials. Nonetheless, a detailed molecular analysis of important protein features, such as the origin of its ion selectivity and the rather low conductance as compared to other channel homologues, has been unfeasible until the recent crystallization of the mouse 5-HT3A receptor. Here, we present extended molecular dynamics simulations and free energy calculations of the whole 5-HT3A protein with the aim of better understanding its ion transport properties, such as the pathways for ion permeation into the receptor body and the complex nature of the selectivity filter. Our investigation unravels previously unpredicted structural features of the 5-HT3A receptor, such as the existence of alternative intersubunit pathways for ion translocation at the interface between the extracellular and the transmembrane domains, in addition to the one along the channel main axis. Moreover, our study offers a molecular interpretation of the role played by an arginine triplet located in the intracellular domain on determining the characteristic low conductance of the 5-HT3A receptor, as evidenced in previous experiments. In view of these results, possible implications on other members of the superfamily are suggested. PMID:26465896

  3. Pathways and Barriers for Ion Translocation through the 5-HT3A Receptor Channel.

    PubMed

    Di Maio, Danilo; Chandramouli, Balasubramanian; Brancato, Giuseppe

    2015-01-01

    Pentameric ligand gated ion channels (pLGICs) are ionotropic receptors that mediate fast intercellular communications at synaptic level and include either cation selective (e.g., nAChR and 5-HT3) or anion selective (e.g., GlyR, GABAA and GluCl) membrane channels. Among others, 5-HT3 is one of the most studied members, since its first cloning back in 1991, and a large number of studies have successfully pinpointed protein residues critical for its activation and channel gating. In addition, 5-HT3 is also the target of a few pharmacological treatments due to the demonstrated benefits of its modulation in clinical trials. Nonetheless, a detailed molecular analysis of important protein features, such as the origin of its ion selectivity and the rather low conductance as compared to other channel homologues, has been unfeasible until the recent crystallization of the mouse 5-HT3A receptor. Here, we present extended molecular dynamics simulations and free energy calculations of the whole 5-HT3A protein with the aim of better understanding its ion transport properties, such as the pathways for ion permeation into the receptor body and the complex nature of the selectivity filter. Our investigation unravels previously unpredicted structural features of the 5-HT3A receptor, such as the existence of alternative intersubunit pathways for ion translocation at the interface between the extracellular and the transmembrane domains, in addition to the one along the channel main axis. Moreover, our study offers a molecular interpretation of the role played by an arginine triplet located in the intracellular domain on determining the characteristic low conductance of the 5-HT3A receptor, as evidenced in previous experiments. In view of these results, possible implications on other members of the superfamily are suggested. PMID:26465896

  4. Fluorophore assisted light inactivation (FALI) of recombinant 5-HT3A receptor constitutive internalization and function

    PubMed Central

    Morton, Russell A.; Luo, Guoxiang; Davis, Margaret I.; Hales, Tim G.; Lovinger, David M.

    2011-01-01

    Fluorescent proteins and molecules are now widely used to tag and visualize proteins resulting in an improved understanding of protein trafficking, localization, and function. In addition, fluorescent tags have also been used to inactivate protein function in a spatially and temporally-defined manner, using a technique known as fluorophore-assisted light inactivation (FALI) or chromophore-assisted light inactivation (CALI). In this study we tagged the serotonin3 A subunit with the α-bungarotoxin binding sequence (BBS) and subsequently labeled 5-HT3A/BBS receptors with fluorescently conjugated α-bungarotoxin in live cells. We show that 5-HT3A/BBS receptors are constitutively internalized in the absence of an agonist and internalization as well as receptor function are inhibited by fluorescence. The fluorescence-induced disruption of function and internalization was reduced with oxygen radical scavengers suggesting the involvement of reactive oxygen species, implicating the FALI process. Furthermore, these data suggest that intense illumination during live-cell microscopy may result in inadvertent FALI and inhibition of protein trafficking. PMID:21338684

  5. Influence of the 5-HT3A Receptor Gene Polymorphism and Childhood Sexual Trauma on Central Serotonin Activity

    PubMed Central

    Huh, Hyu Jung; Chae, Jeong-Ho

    2015-01-01

    Background Gene-environment interactions are important for understanding alterations in human brain function. The loudness dependence of auditory evoked potential (LDAEP) is known to reflect central serotonergic activity. Single nucleotide polymorphisms (SNPs) in the 5-HT3A serotonin receptor gene are associated with psychiatric disorders. This study aimed to investigate the effect between 5-HT3A receptor gene polymorphisms and childhood sexual trauma on the LDAEP as an electrophysiological marker in healthy subjects. Methods A total of 206 healthy subjects were recruited and evaluated using the childhood trauma questionnaire (CTQ) and hospital anxiety and depression scale (HADS). Peak-to-peak N1/P2 was measured at five stimulus intensities, and the LDAEP was calculated as the linear-regression slope. In addition, the rs1062613 SNPs of 5-HT3A (CC, CT, and TT) were analyzed in healthy subjects. Results There was a significant interaction between scores on the CTQ-sexual abuse subscale and 5-HT3A genotype on the LDAEP. Subjects with the CC polymorphism had a significantly higher LDEAP than T carriers in the sexually abused group. In addition, CC genotype subjects in the sexually abused group showed a significantly higher LDAEP compared with CC genotype subjects in the non-sexually abused group. Conclusions Our findings suggest that people with the CC polymorphism of the 5-HT3A gene have a greater risk of developing mental health problems if they have experienced childhood sexual abuse, possibly due to low central serotonin activity. Conversely, the T polymorphism may be protective against any central serotonergic changes following childhood sexual trauma. PMID:26701104

  6. Kampo Medicine: Evaluation of the Pharmacological Activity of 121 Herbal Drugs on GABAA and 5-HT3A Receptors.

    PubMed

    Hoffmann, Katrin M; Herbrechter, Robin; Ziemba, Paul M; Lepke, Peter; Beltrán, Leopoldo; Hatt, Hanns; Werner, Markus; Gisselmann, Günter

    2016-01-01

    Kampo medicine is a form of Japanese phytotherapy originating from traditional Chinese medicine (TCM). During the last several decades, much attention has been paid to the pharmacological effects of these medical plants and their constituents. However, in many cases, a systematic screening of Kampo remedies to determine pharmacologically relevant targets is still lacking. In this study, a broad screening of Kampo remedies was performed to look for pharmacologically relevant 5-HT3A and GABAA receptor ligands. Several of the Kampo remedies are currently used for symptoms such as nausea, emesis, gastrointestinal motility disorders, anxiety, restlessness, or insomnia. Therefore, the pharmacological effects of 121 herbal drugs from Kampo medicine were analyzed as ethanol tinctures on heterologously expressed 5-HT3A and GABAA receptors, due to the involvement of these receptors in such pathophysiological processes. The tinctures of Lindera aggregata (radix) and Leonurus japonicus (herba) were the most effective inhibitory compounds on the 5-HT3A receptor. Further investigation of known ingredients in these compounds led to the identification of leonurine from Leonurus as a new natural 5-HT3A receptor antagonist. Several potentiating herbs (e.g., Magnolia officinalis (cortex), Syzygium aromaticum (flos), and Panax ginseng (radix)) were also identified for the GABAA receptor, which are all traditionally used for their sedative or anxiolytic effects. A variety of tinctures with antagonistic effects Salvia miltiorrhiza (radix) were also detected. Therefore, this study reveals new insights into the pharmacological action of a broad spectrum of herbal drugs from Kampo, allowing for a better understanding of their physiological effects and clinical applications. PMID:27524967

  7. Kampo Medicine: Evaluation of the Pharmacological Activity of 121 Herbal Drugs on GABAA and 5-HT3A Receptors

    PubMed Central

    Hoffmann, Katrin M.; Herbrechter, Robin; Ziemba, Paul M.; Lepke, Peter; Beltrán, Leopoldo; Hatt, Hanns; Werner, Markus; Gisselmann, Günter

    2016-01-01

    Kampo medicine is a form of Japanese phytotherapy originating from traditional Chinese medicine (TCM). During the last several decades, much attention has been paid to the pharmacological effects of these medical plants and their constituents. However, in many cases, a systematic screening of Kampo remedies to determine pharmacologically relevant targets is still lacking. In this study, a broad screening of Kampo remedies was performed to look for pharmacologically relevant 5-HT3A and GABAA receptor ligands. Several of the Kampo remedies are currently used for symptoms such as nausea, emesis, gastrointestinal motility disorders, anxiety, restlessness, or insomnia. Therefore, the pharmacological effects of 121 herbal drugs from Kampo medicine were analyzed as ethanol tinctures on heterologously expressed 5-HT3A and GABAA receptors, due to the involvement of these receptors in such pathophysiological processes. The tinctures of Lindera aggregata (radix) and Leonurus japonicus (herba) were the most effective inhibitory compounds on the 5-HT3A receptor. Further investigation of known ingredients in these compounds led to the identification of leonurine from Leonurus as a new natural 5-HT3A receptor antagonist. Several potentiating herbs (e.g., Magnolia officinalis (cortex), Syzygium aromaticum (flos), and Panax ginseng (radix)) were also identified for the GABAA receptor, which are all traditionally used for their sedative or anxiolytic effects. A variety of tinctures with antagonistic effects Salvia miltiorrhiza (radix) were also detected. Therefore, this study reveals new insights into the pharmacological action of a broad spectrum of herbal drugs from Kampo, allowing for a better understanding of their physiological effects and clinical applications. PMID:27524967

  8. Investigation of 5-HT3A receptor gene expression in peripheral blood mononuclear cells of individuals who had been exposed to air pollution.

    PubMed

    Ahangari, Ghasem; Amirabad, Leila Mohammadi; Mozafari, Sona; Majeidi, Ali; Deilami, Gholamreza Derkhshan

    2013-12-01

    The role of air pollution in exacerbation of allergic symptoms is well known. Several studies have shown the effect of air pollution on serotonergic system. The changes in serotonergic system could trigger several allergic symptoms. 5-HT(3A) is among serotonin receptors on the peripheral Blood Mononuclear Cells (PBMCs) as well as other cells. In the present study we compared the 5-HT(3A) gene expression in PBMCs of the asthmatic patients as well as individuals who had been exposed to the air pollution. Normal individuals were also included in the study as control for comparison of 5-HT(3A) gene expression. Following the synthesis of the cDNA using mRNA extracted from PBMCs the level of 5- HT(3A) gene expression was measured using real-time PCR. The results showed t a significant increase in the relative expression level of 5-HT(3A) receptor in PBMCs from asthmatic patients and individuals exposed to the air pollutants compared to normal controls. Our result indicates that significant increase in 5-HT(3A) receptor may contribute to the pathogenesis as well as allergic symptoms which resulted from air pollution.

  9. Length and Amino Acid Sequence of Peptides Substituted for the 5-HT3A Receptor M3M4 Loop May Affect Channel Expression and Desensitization

    PubMed Central

    McKinnon, Nicole K.; Bali, Moez; Akabas, Myles H.

    2012-01-01

    5-HT3A receptors are pentameric neurotransmitter-gated ion channels in the Cys-loop receptor family. Each subunit contains an extracellular domain, four transmembrane segments (M1, M2, M3, M4) and a 115 residue intracellular loop between M3 and M4. In contrast, the M3M4 loop in prokaryotic homologues is <15 residues. To investigate the limits of M3M4 loop length and composition on channel function we replaced the 5-HT3A M3M4 loop with two to seven alanine residues (5-HT3A-An = 2–7). Mutants were expressed in Xenopus laevis oocytes and characterized using two electrode voltage clamp recording. All mutants were functional. The 5-HT EC50's were at most 5-fold greater than wild-type (WT). The desensitization rate differed significantly among the mutants. Desensitization rates for 5-HT3A-A2, 5-HT3A-A4, 5-HT3A-A6, and 5-HT3A-A7 were similar to WT. In contrast, 5-HT3A-A3 and 5-HT3A-A5 had desensitization rates at least an order of magnitude faster than WT. The one Ala loop construct, 5-HT3A-A1, entered a non-functional state from which it did not recover after the first 5-HT application. These results suggest that the large M3M4 loop of eukaryotic Cys-loop channels is not required for receptor assembly or function. However, loop length and amino acid composition can effect channel expression and desensitization. We infer that the cytoplasmic ends of the M3 and M4 segments may undergo conformational changes during channel gating and desensitization and/or the loop may influence the position and mobility of these segments as they undergo gating-induced conformational changes. Altering structure or conformational mobility of the cytoplasmic ends of M3 and M4 may be the basis by which phosphorylation or protein binding to the cytoplasmic loop alters channel function. PMID:22539982

  10. Regulation of the 5-HT3A receptor-mediated current by alkyl 4-hydroxybenzoates isolated from the seeds of Nelumbo nucifera.

    PubMed

    Youn, Ui Joung; Lee, Jun-Ho; Lee, Yoo Jin; Nam, Joo Won; Bae, Hyunsu; Seo, Eun-Kyoung

    2010-09-01

    Four known alkyl 4-hydroxybenzoates, i.e., methyl 4-hydroxybenzoate (1), ethyl 4-hydroxybenzoate (2), propyl 4-hydroxybenzoate (3), and butyl 4-hydroxybenzoate (4), were isolated from the seeds of Nelumbo nucifera Gaertner (Nymphaeaceae) for the first time. The structures of the isolates were identified by 1D- and 2D-NMR spectroscopy and comparison with published values. The compounds were evaluated for their effects on the 5-HT-stimulated inward current (I(5-HT)) mediated by the human 5-HT(3)A receptors expressed in Xenopus oocytes. Compounds 1 and 2 enhanced the I(5-HT), but 4 reduced it. These results indicate that 4 is an inhibitor of the 5-HT(3)A receptors expressed in Xenopus oocytes.

  11. Regulation of the 5-HT3A receptor-mediated current by alkyl 4-hydroxybenzoates isolated from the seeds of Nelumbo nucifera.

    PubMed

    Youn, Ui Joung; Lee, Jun-Ho; Lee, Yoo Jin; Nam, Joo Won; Bae, Hyunsu; Seo, Eun-Kyoung

    2010-09-01

    Four known alkyl 4-hydroxybenzoates, i.e., methyl 4-hydroxybenzoate (1), ethyl 4-hydroxybenzoate (2), propyl 4-hydroxybenzoate (3), and butyl 4-hydroxybenzoate (4), were isolated from the seeds of Nelumbo nucifera Gaertner (Nymphaeaceae) for the first time. The structures of the isolates were identified by 1D- and 2D-NMR spectroscopy and comparison with published values. The compounds were evaluated for their effects on the 5-HT-stimulated inward current (I(5-HT)) mediated by the human 5-HT(3)A receptors expressed in Xenopus oocytes. Compounds 1 and 2 enhanced the I(5-HT), but 4 reduced it. These results indicate that 4 is an inhibitor of the 5-HT(3)A receptors expressed in Xenopus oocytes. PMID:20860031

  12. Serotonin homeostasis and serotonin receptors as actors of cortical construction: special attention to the 5-HT3A and 5-HT6 receptor subtypes

    PubMed Central

    Vitalis, Tania; Ansorge, Mark S.; Dayer, Alexandre G.

    2013-01-01

    Cortical circuits control higher-order cognitive processes and their function is highly dependent on their structure that emerges during development. The construction of cortical circuits involves the coordinated interplay between different types of cellular processes such as proliferation, migration, and differentiation of neural and glial cell subtypes. Among the multiple factors that regulate the assembly of cortical circuits, 5-HT is an important developmental signal that impacts on a broad diversity of cellular processes. 5-HT is detected at the onset of embryonic telencephalic formation and a variety of serotonergic receptors are dynamically expressed in the embryonic developing cortex in a region and cell-type specific manner. Among these receptors, the ionotropic 5-HT3A receptor and the metabotropic 5-HT6 receptor have recently been identified as novel serotonergic targets regulating different aspects of cortical construction including neuronal migration and dendritic differentiation. In this review, we focus on the developmental impact of serotonergic systems on the construction of cortical circuits and discuss their potential role in programming risk for human psychiatric disorders. PMID:23801939

  13. 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 ...

  14. Design and validation of a homogeneous time-resolved fluorescence cell-based assay targeting the ligand-gated ion channel 5-HT3A.

    PubMed

    Blanc, Emilie; Wagner, Patrick; Plaisier, Fabrice; Schmitt, Martine; Durroux, Thierry; Bourguignon, Jean-Jacques; Partiseti, Michel; Dupuis, Elodie; Bihel, Frederic

    2015-09-01

    Ligand-gated ion channels (LGICs) are considered as attractive protein targets in the search for new therapeutic agents. Nowadays, this strategy involves the capability to screen large chemical libraries. We present a new Tag-lite ligand binding assay targeting LGICs on living cells. This technology combines the use of suicide enzyme tags fused to channels of interest with homogeneous time-resolved fluorescence (HTRF) as the detection readout. Using the 5-HT3 receptor as system model, we showed that the pharmacology of the HALO-5HT3 receptor was identical to that of the native receptor. After validation of the assay by using 5-HT3 agonists and antagonists of reference, a pilot screen enabled us to identify azelastine, a well-known histamine H1 antagonist, as a potent 5-HT3 antagonist. This interesting result was confirmed with electrophysiological experiments. The method described here is easy to implement and could be applicable for other LGICs, opening new ways for the screening of chemical libraries. PMID:25998104

  15. 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.

  16. 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

  17. 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

  18. 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.

  19. 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...

  20. Effects of D1 receptor knockout on fear and reward learning.

    PubMed

    Abraham, Antony D; Neve, Kim A; Lattal, K Matthew

    2016-09-01

    Dopamine signaling is involved in a variety of neurobiological processes that contribute to learning and memory. D1-like dopamine receptors (including D1 and D5 receptors) are thought to be involved in memory and reward processes, but pharmacological approaches have been limited in their ability to distinguish between D1 and D5 receptors. Here, we examine the effects of a specific knockout of D1 receptors in associative learning tasks involving aversive (shock) or appetitive (cocaine) unconditioned stimuli. We find that D1 knockout mice show similar levels of cued and contextual fear conditioning to WT controls following conditioning protocols involving one, two, or four shocks. D1 knockout mice show increased generalization of fear conditioning and extinction across contexts, revealed as increased freezing to a novel context following conditioning and decreased freezing to an extinguished cue during a contextual renewal test. Further, D1 knockout mice show mild enhancements in extinction following an injection of SKF81297, a D1/D5 receptor agonist, suggesting a role for D5 receptors in extinction enhancements induced by nonspecific pharmacological agonists. Finally, although D1 knockout mice show decreased locomotion induced by cocaine, they are able to form a cocaine-induced conditioned place preference. We discuss these findings in terms of the role of dopamine D1 receptors in general learning and memory processes. PMID:27423521

  1. Rescue of the mineralocorticoid receptor knock-out mouse.

    PubMed

    Bleich, M; Warth, R; Schmidt-Hieber, M; Schulz-Baldes, A; Hasselblatt, P; Fisch, D; Berger, S; Kunzelmann, K; Kriz, W; Schütz, G; Greger, R

    1999-08-01

    The mineralocorticoid receptor knock-out mouse (MR-/-), resembling inborn pseudohypoaldosteronism, dies 8-12 days after birth in circulatory failure with all the signs of terminal volume contraction. The present study aimed to examine the functional defects in the kidney and colon in detail and to attempt to rescue these mice. In neonatal (nn) MR-/- the amiloride-sensitive short-circuit current in the colon was reduced to approximately one-third compared to controls (MR+/+ and MR+/-). In isolated in vitro perfused collecting ducts the amiloride-induced hyperpolarization of the basolateral membrane (Vbl) of nn MR-/- was similar to that of controls, but urinary Na+ excretion was markedly increased to 4.3 micromol/day.g (BW). Based on this measured urinary Na+ loss we tried to rescue nn MR-/- mice by injecting NaCl twice daily (3.85 micromol/g BW), corresponding to 22 microliter of isotonic saline/g BW subcutaneously. This regimen was continued until the animals had reached a body mass of 8.5 g. Thereafter, in addition to normal chow and tap water, NaCl drinking water (333 mmol/l) and pellets soaked in 333 mmol/l NaCl were offered. Unlike the untreated nn MR-/- most of these mice survived. The adult animals were examined between days 27 and 41, some were used for breeding. When compared to age-matched controls the growth of MR-/- was delayed until day 20. Then their growth curve increased in slope and reached that of controls. MR-/- retained their Na+-losing defect. Amiloride's effect on urinary Na+ excretion was not significant in MR-/- mice and the effect on Vbl in isolated cortical collecting ducts was attenuated. The renin-producing cells were hypertrophic and hyperplastic. Plasma renin and aldosterone concentrations were significantly elevated in MR-/- mice. These data indicate that MR-/- can be rescued by timely and matched NaCl substitutions. This enables the animals to develop through a critical phase of life, after which they adapt their oral salt and water

  2. Enhanced sexual behaviors and androgen receptor immunoreactivity in the male progesterone receptor knockout mouse.

    PubMed

    Schneider, Johanna S; Burgess, Carly; Sleiter, Nicole C; DonCarlos, Lydia L; Lydon, John P; O'Malley, Bert; Levine, Jon E

    2005-10-01

    Reproductive and behavioral functions of progesterone receptors (PRs) in males were assessed by examining consequences of PR gene deletion. Basal hormone levels were measured in male progesterone receptor knockout (PRKO) mice and compared to wild-type (WT) counterparts. RIA of serum LH, testosterone, and progesterone levels revealed no significant differences. Levels of FSH were moderately but significantly lower and inhibin levels were higher in PRKOs; these differences were not accompanied by gross differences in testicular weight or morphology. PRKOs exhibited significant alterations in sexual behavior. In initial tests PRKOs exhibited reduced latency to mount, compared with WT. In second sessions, PRKOs again showed a significantly reduced latency to mount and increased likelihood of achieving ejaculation. RU486 treatment in WT produced increased mount and intromission frequency and decreased latency to intromission. In anxiety-related behavior tests, PRKO mice exhibited intermediate anxiety levels, compared with WT, suggesting that enhanced sexual behavior in PRKOs is not secondary to reduced anxiety. Immunohistochemical analysis revealed significantly enhanced androgen receptor expression in the medial preoptic nucleus and bed nucleus of the stria terminalis of PRKO. We conclude that testicular development and function and homeostatic regulation of the hypothalamic-pituitary testicular axis are altered to a lesser extent by PR gene deletion. In contrast, PR appears to play a substantial role in inhibiting the anticipatory/motivational components of male sexual behavior in the mouse. The biological significance of this inhibitory mechanism and the extent to which it is mediated by reduced androgen receptor expression remain to be clarified.

  3. Normal maternal behavior, but increased pup mortality, in conditional oxytocin receptor knockout females.

    PubMed

    Macbeth, Abbe H; Stepp, Jennifer E; Lee, Heon-Jin; Young, W Scott; Caldwell, Heather K

    2010-10-01

    Oxytocin (Oxt) and the Oxt receptor (Oxtr) are implicated in the onset of maternal behavior in a variety of species. Recently, we developed two Oxtr knockout lines: a total body knockout (Oxtr-/-) and a conditional Oxtr knockout (OxtrFB/FB) in which the Oxtr is lacking only in regions of the forebrain, allowing knockout females to potentially nurse and care for their biological offspring. In the current study, we assessed maternal behavior of postpartum OxtrFB/FB females toward their own pups and maternal behavior of virgin Oxtr-/- females toward foster pups and compared knockouts of both lines to wildtype (Oxtr+/+) littermates. We found that both Oxtr-/- and OxtrFB/FB females appear to have largely normal maternal behaviors. However, with first litters, approximately 40% of the OxtrFB/FB knockout dams experienced high pup mortality, compared to fewer than 10% of the Oxtr+/+ dams. We then went on to test whether or not this phenotype occurred in subsequent litters or when the dams were exposed to an environmental disturbance. We found that regardless of the degree of external disturbance, OxtrFB/FB females lost more pups on their first and second litters compared to wildtype females. Possible reasons for higher pup mortality in OxtrFB/FB females are discussed.

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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

  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. 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

  11. Quantification of beta adrenergic receptor subtypes in beta-arrestin knockout mouse airways.

    PubMed

    Hegde, Akhil; Strachan, Ryan T; Walker, Julia K L

    2015-01-01

    In allergic asthma Beta 2 adrenergic receptors (β2ARs) are important mediators of bronchorelaxation and, paradoxically, asthma development. This contradiction is likely due to the activation of dual signaling pathways that are downstream of G proteins or β-arrestins. Our group has recently shown that β-arrestin-2 acts in its classical role to desensitize and constrain β2AR-induced relaxation of both human and murine airway smooth muscle. To assess the role of β-arrestins in regulating β2AR function in asthma, we and others have utilized β-arrestin-1 and -2 knockout mice. However, it is unknown if genetic deletion of β-arrestins in these mice influences β2AR expression in the airways. Furthermore, there is lack of data on compensatory expression of βAR subtypes when either of the β-arrestins is genetically deleted, thus necessitating a detailed βAR subtype expression study in these β-arrestin knockout mice. Here we standardized a radioligand binding methodology to characterize and quantitate βAR subtype distribution in the airway smooth muscle of wild-type C57BL/6J and β-arrestin-1 and β-arrestin-2 knockout mice. Using complementary competition and single-point saturation binding assays we found that β2ARs predominate over β1ARs in the whole lung and epithelium-denuded tracheobronchial smooth muscle of C57BL/6J mice. Quantification of βAR subtypes in β-arrestin-1 and β-arrestin-2 knockout mouse lung and epithelium-denuded tracheobronchial tissue showed that, similar to the C57BL/6J mice, both knockouts display a predominance of β2AR expression. These data provide further evidence that β2ARs are expressed in greater abundance than β1ARs in the tracheobronchial smooth muscle and that loss of either β-arrestin does not significantly affect the expression or relative proportions of βAR subtypes. As β-arrestins are known to modulate β2AR function, our analysis of βAR subtype expression in β-arrestin knockout mice airways sets a reference

  12. 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

  13. 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.

  14. HSL-knockout mouse testis exhibits class B scavenger receptor upregulation and disrupted lipid raft microdomains.

    PubMed

    Casado, María Emilia; Huerta, Lydia; Ortiz, Ana Isabel; Pérez-Crespo, Mirian; Gutiérrez-Adán, Alfonso; Kraemer, Fredric B; Lasunción, Miguel Ángel; Busto, Rebeca; Martín-Hidalgo, Antonia

    2012-12-01

    There is a tight relationship between fertility and changes in cholesterol metabolism during spermatogenesis. In the testis, class B scavenger receptors (SR-B) SR-BI, SR-BII, and LIMP II mediate the selective uptake of cholesterol esters from HDL, which are hydrolyzed to unesterified cholesterol by hormone-sensitive lipase (HSL). HSL is critical because HSL knockout (KO) male mice are sterile. The aim of the present work was to determine the effects of the lack of HSL in testis on the expression of SR-B, lipid raft composition, and related cell signaling pathways. HSL-KO mouse testis presented altered spermatogenesis associated with decreased sperm counts, sperm motility, and infertility. In wild-type (WT) testis, HSL is expressed in elongated spermatids; SR-BI, in Leydig cells and spermatids; SR-BII, in spermatocytes and spermatids but not in Leydig cells; and LIMP II, in Sertoli and Leydig cells. HSL knockout male mice have increased expression of class B scavenger receptors, disrupted caveolin-1 localization in lipid raft plasma membrane microdomains, and activated phospho-ERK, phospho-AKT, and phospho-SRC in the testis, suggesting that class B scavenger receptors are involved in cholesterol ester uptake for steroidogenesis and spermatogenesis in the testis.

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. 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

  1. Effect of P2X7 Receptor Knockout on AQP-5 Expression of Type I Alveolar Epithelial Cells

    PubMed Central

    Ebeling, Georg; Bläsche, Robert; Hofmann, Falk; Augstein, Antje; Kasper, Michael; Barth, Kathrin

    2014-01-01

    P2X7 receptors, ATP-gated cation channels, are specifically expressed in alveolar epithelial cells. The pathophysiological function of this lung cell type, except a recently reported putative involvement in surfactant secretion, is unknown. In addition, P2X7 receptor-deficient mice show reduced inflammation and lung fibrosis after exposure with bleomycin. To elucidate the role of the P2X7 receptor in alveolar epithelial type I cells we characterized the pulmonary phenotype of P2X7 receptor knockout mice by using immunohistochemistry, western blot analysis and real-time RT PCR. No pathomorphological signs of fibrosis were found. Results revealed, however, a remarkable loss of aquaporin-5 protein and mRNA in young knockout animals. Additional in vitro experiments with bleomycin treated precision cut lung slices showed a greater sensitivity of the P2X7 receptor knockout mice in terms of aquaporin-5 reduction as wild type animals. Finally, P2X7 receptor function was examined by using the alveolar epithelial cell lines E10 and MLE-12 for stimulation experiments with bleomycin. The in vitro activation of P2X7 receptor was connected with an increase of aquaporin-5, whereas the inhibition of the receptor with oxidized ATP resulted in down regulation of aquaporin-5. The early loss of aquaporin-5 which can be found in different pulmonary fibrosis models does not implicate a specific pathogenetic role during fibrogenesis. PMID:24941004

  2. 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

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. [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.

  8. 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.

  9. Knockout of NMDA receptors in parvalbumin interneurons recreates autism-like phenotypes.

    PubMed

    Saunders, John A; Tatard-Leitman, Valerie M; Suh, Jimmy; Billingslea, Eddie N; Roberts, Timothy P; Siegel, Steven J

    2013-04-01

    Autism is a disabling neurodevelopmental disorder characterized by social deficits, language impairment, and repetitive behaviors with few effective treatments. New evidence suggests that autism has reliable electrophysiological endophenotypes and that these measures may be caused by n-methyl-d-aspartic acid receptor (NMDAR) disruption on parvalbumin (PV)-containing interneurons. These findings could be used to create new translational biomarkers. Recent developments have allowed for cell-type selective knockout of NMDARs in order to examine the perturbations caused by disrupting specific circuits. This study examines several electrophysiological and behavioral measures disrupted in autism using a PV-selective reduction in NMDA R1 subunit. Mouse electroencephalograph (EEG) was recorded in response to auditory stimuli. Event-related potential (ERP) component amplitude and latency analysis, social testing, and premating ultrasonic vocalizations (USVs) recordings were performed. Correlations were examined between the ERP latency and behavioral measures. The N1 ERP latency was delayed, sociability was reduced, and mating USVs were impaired in PV-selective NMDA Receptor 1 Knockout (NR1 KO) as compared with wild-type mice. There was a significant correlation between N1 latency and sociability but not between N1 latency and premating USV power or T-maze performance. The increases in N1 latency, impaired sociability, and reduced vocalizations in PV-selective NR1 KO mice mimic similar changes found in autism. Electrophysiological changes correlate to reduced sociability, indicating that the local circuit mechanisms controlling N1 latency may be utilized in social function. Therefore, we propose that behavioral and electrophysiological alterations in PV-selective NR1 KO mice may serve as a useful model for therapeutic development in autism. Autism Res 2013, 6: 69-77. © 2013 International Society for Autism Research, Wiley Periodicals, Inc.

  10. Conditional Knockout of NMDA Receptors in Dopamine Neurons Prevents Nicotine-Conditioned Place Preference

    PubMed Central

    Phillip Wang, Lei; Li, Fei; Shen, Xiaoming; Tsien, Joe Z.

    2010-01-01

    Nicotine from smoking tobacco produces one of the most common forms of addictive behavior and has major societal and health consequences. It is known that nicotine triggers tobacco addiction by activating nicotine acetylcholine receptors (nAChRs) in the midbrain dopaminergic reward system, primarily via the ventral tegmental area. Heterogeneity of cell populations in the region has made it difficult for pharmacology-based analyses to precisely assess the functional significance of glutamatergic inputs to dopamine neurons in nicotine addiction. By generating dopamine neuron-specific NR1 knockout mice using cre/loxP-mediated method, we demonstrate that genetic inactivation of the NMDA receptors in ventral tegmental area dopamine neurons selectively prevents nicotine-conditioned place preference. Interestingly, the mutant mice exhibit normal performances in the conditioned place aversion induced by aversive air puffs. Therefore, this selective effect on addictive drug-induced reinforcement behavior suggests that NMDA receptors in the dopamine neurons are critical for the development of nicotine addiction. PMID:20062537

  11. Brain Region-Specific Effects of cGMP-Dependent Kinase II Knockout on AMPA Receptor Trafficking and Animal Behavior

    ERIC Educational Resources Information Center

    Kim, Seonil; Pick, Joseph E.; Abera, Sinedu; Khatri, Latika; Ferreira, Danielle D. P.; Sathler, Matheus F.; Morison, Sage L.; Hofmann, Franz; Ziff, Edward B.

    2016-01-01

    Phosphorylation of GluA1, a subunit of AMPA receptors (AMPARs), is critical for AMPAR synaptic trafficking and control of synaptic transmission. cGMP-dependent protein kinase II (cGKII) mediates this phosphorylation, and cGKII knockout (KO) affects GluA1 phosphorylation and alters animal behavior. Notably, GluA1 phosphorylation in the KO…

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. Acceleration of atherogenesis by COX-1-dependent prostanoid formation in low density lipoprotein receptor knockout mice.

    PubMed

    Praticò, D; Tillmann, C; Zhang, Z B; Li, H; FitzGerald, G A

    2001-03-13

    The cyclooxygenase (COX) product, prostacyclin (PGI(2)), inhibits platelet activation and vascular smooth-muscle cell migration and proliferation. Biochemically selective inhibition of COX-2 reduces PGI(2) biosynthesis substantially in humans. Because deletion of the PGI(2) receptor accelerates atherogenesis in the fat-fed low density lipoprotein receptor knockout mouse, we wished to determine whether selective inhibition of COX-2 would accelerate atherogenesis in this model. To address this hypothesis, we used dosing with nimesulide, which inhibited COX-2 ex vivo, depressed urinary 2,3 dinor 6-keto PGF(1alpha) by approximately 60% but had no effect on thromboxane formation by platelets, which only express COX-1. By contrast, the isoform nonspecific inhibitor, indomethacin, suppressed platelet function and thromboxane formation ex vivo and in vivo, coincident with effects on PGI(2) biosynthesis indistinguishable from nimesulide. Indomethacin reduced the extent of atherosclerosis by 55 +/- 4%, whereas nimesulide failed to increase the rate of atherogenesis. Despite their divergent effects on atherogenesis, both drugs depressed two indices of systemic inflammation, soluble intracellular adhesion molecule-1, and monocyte chemoattractant protein-1 to a similar but incomplete degree. Neither drug altered serum lipids and the marked increase in vascular expression of COX-2 during atherogenesis. Accelerated progression of atherosclerosis is unlikely during chronic intake of specific COX-2 inhibitors. Furthermore, evidence that COX-1-derived prostanoids contribute to atherogenesis suggests that controlled evaluation of the effects of nonsteroidal anti-inflammatory drugs and/or aspirin on plaque progression in humans is timely.

  17. 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.

  18. Dysregulation of Uterine Signaling Pathways in Progesterone Receptor-Cre Knockout of Dicer

    PubMed Central

    Andreu-Vieyra, Claudia V.; Kim, Tae Hoon; Jeong, Jae-Wook; Hodgson, Myles C.; Chen, Ruihong; Creighton, Chad J.; Lydon, John P.; Gunaratne, Preethi H.; DeMayo, Francesco J.; Matzuk, Martin M.

    2012-01-01

    Epithelial-stromal interactions in the uterus are required for normal uterine functions such as pregnancy, and multiple signaling pathways are essential for this process. Although Dicer and microRNA (miRNA) have been implicated in several reproductive processes, the specific roles of Dicer and miRNA in uterine development are not known. To address the roles of miRNA in the regulation of key uterine pathways, we generated a conditional knockout of Dicer in the postnatal uterine epithelium and stroma using progesterone receptor-Cre. These Dicer conditional knockout females are sterile with small uteri, which demonstrate significant defects, including absence of glandular epithelium and enhanced stromal apoptosis, beginning at approximately postnatal d 15, with coincident expression of Cre and deletion of Dicer. Specific miRNA (miR-181c, −200b, −101, let-7d) were down-regulated and corresponding predicted proapoptotic target genes (Bcl2l11, Aldh1a3) were up-regulated, reflecting the apoptotic phenomenon. Although these mice had normal serum hormone levels, critical uterine signaling pathways, including progesterone-responsive genes, Indian hedgehog signaling, and the Wnt/β-catenin canonical pathway, were dysregulated at the mRNA level. Importantly, uterine stromal cell proliferation in response to progesterone was absent, whereas uterine epithelial cell proliferation in response to estradiol was maintained in adult uteri. These data implicate Dicer and appropriate miRNA expression as essential players in the regulation of multiple uterine signaling pathways required for uterine development and appropriate function. PMID:22798293

  19. 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

  20. The in vivo respiratory phenotype of the adenosine A1 receptor knockout mouse.

    PubMed

    Heitzmann, Dirk; Buehler, Philipp; Schweda, Frank; Georgieff, Michael; Warth, Richard; Thomas, Joerg

    2016-02-01

    The nucleoside adenosine has been implicated in the regulation of respiration, especially during hypoxia in the newborn. In this study the role of adenosine A1 receptors for the control of respiration was investigated in vivo. To this end, respiration of unrestrained adult and neonatal adenosine A1 receptor knockout mice (A1R(-/-)) was measured in a plethysmographic device. Under control conditions (21% O2) and mild hypoxia (12-15% O2) no difference of respiratory parameters was observed between adult wildtype (A1R(+/+)) and A1R(-/-) mice. Under more severe hypoxia (6-10% O2) A1R(+/+) mice showed, after a transient increase of respiration, a decrease of respiration frequency (fR) and tidal volume (VT) leading to a decrease of minute volume (MV). This depression of respiration during severe hypoxia was absent in A1R(-/-) mice which displayed a stimulated respiration as indicated by the enhancement of MV by some 50-60%. During hypercapnia-hyperoxia (3-10% CO2/97-90 % O2), no obvious differences in respiration of A1R(-/-) and A1R(+/+) was observed. In neonatal mice, the respiratory response to hypoxia was surprisingly similar in both genotypes. However, neonatal A1R(-/-) mice appeared to have more frequently periods of apnea during hypoxia and in the post-hypoxic control period. In conclusion, these data indicate that the adenosine A1 receptor is an important molecular component mediating hypoxic depression in adult mice and it appears to stabilize respiration of neonatal mice. PMID:26593641

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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

  7. GABAB receptors and glucose homeostasis: evaluation in GABAB receptor knockout mice.

    PubMed

    Bonaventura, M M; Catalano, P N; Chamson-Reig, A; Arany, E; Hill, D; Bettler, B; Saravia, F; Libertun, C; Lux-Lantos, V A

    2008-01-01

    GABA has been proposed to inhibit insulin secretion through GABAB receptors (GABABRs) in pancreatic beta-cells. We investigated whether GABABRs participated in the regulation of glucose homeostasis in vivo. The animals used in this study were adult male and female BALB/C mice, mice deficient in the GABAB1 subunit of the GABABR (GABAB(-/-)), and wild types (WT). Blood glucose was measured under fasting/fed conditions and in glucose tolerance tests (GTTs) with a Lifescan Glucose meter, and serum insulin was measured by ELISA. Pancreatic insulin content and islet insulin were released by RIA. Western blots for the GABAB1 subunit in islet membranes and immunohistochemistry for insulin and GABAB1 were performed in both genotypes. BALB/C mice preinjected with Baclofen (GABABR agonist, 7.5 mg/kg ip) presented impaired GTTs and decreased insulin secretion compared with saline-preinjected controls. GABAB(-/-) mice showed fasting and fed glucose levels similar to WT. GABAB(-/-) mice showed improved GTTs at moderate glucose overloads (2 g/kg). Baclofen pretreatment did not modify GTTs in GABAB(-/-) mice, whereas it impaired normal glycemia reinstatement in WT. Baclofen inhibited glucose-stimulated insulin secretion in WT isolated islets but was without effect in GABAB(-/-) islets. In GABAB(-/-) males, pancreatic insulin content was increased, basal and glucose-stimulated insulin secretion were augmented, and impaired insulin tolerance test and increased homeostatic model assessment of insulin resistance index were determined. Immunohistochemistry for insulin demonstrated an increase of very large islets in GABAB(-/-) males. Results demonstrate that GABABRs are involved in the regulation of glucose homeostasis in vivo and that the constitutive absence of GABABRs induces alterations in pancreatic histology, physiology, and insulin resistance. PMID:17971510

  8. 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

  9. Regulation of FSHbeta and GnRH receptor gene expression in activin receptor II knockout male mice.

    PubMed

    Kumar, T Rajendra; Agno, Julio; Janovick, Jo Ann; Conn, P Michael; Matzuk, Martin M

    2003-12-30

    To examine in vivo, the local effects of inhibins and activins within the anterior pituitary, independent of their endocrine effects exerted from the gonad, in mediating FSH homeostasis, we used castrated knockout mice lacking either inhibin alpha or activin receptor II (ACVR2) alone or in combination. Compared to castrated wild-type (WT) mice, FSHbeta mRNA levels in the pituitaries of Acvr2 null mice were significantly downregulated in the absence of gonadal feedback. FSHbeta mRNA levels were not significantly higher in the pituitaries of castrated inhibin alpha null mice compared to those in Acvr2 null mice and remained the same in the pituitaries of castrated double mutant mice lacking both inhibin and ACVR2. In contrast to FSHbeta mRNA expression changes, pituitary FSH content was significantly reduced in Acvr2 null mice whereas it was only slightly upregulated in inhibin alpha null mice. Combined absence of both ACVR2 signaling and inhibins caused a decrease in FSH content compared to that in the absence of inhibins alone. These changes in pituitary content were in parallel to those in serum FSH levels in these three groups of castrated mice, suggesting that the unopposed actions of locally produced inhibins are dominant over those effects mediated by ACVR2 signaling to regulate FSH biosynthesis and secretion. Thus, our in vivo results demonstrate that within the pituitary, locally produced activins and inhibins exert their actions at distinct phases of FSH homeostasis. In an independent set of experiments, we tested whether in vivo signaling via ACVR2 is necessary for hypothalamic GnRH biosynthesis and for GnRH receptor expression. Our results demonstrate that in contrast to previous in vitro studies, signaling through ACVR2 is neither required for hypothalamic synthesis of GnRH peptide nor for expression of GnRH receptors in the anterior pituitary. We conclude that within the hypothalamic-pituitary short loop, ACVR2 signaling is critical only for FSH

  10. 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.

  11. A satellite cell-specific knockout of the androgen receptor reveals myostatin as a direct androgen target in skeletal muscle.

    PubMed

    Dubois, Vanessa; Laurent, Michaël R; Sinnesael, Mieke; Cielen, Nele; Helsen, Christine; Clinckemalie, Liesbeth; Spans, Lien; Gayan-Ramirez, Ghislaine; Deldicque, Louise; Hespel, Peter; Carmeliet, Geert; Vanderschueren, Dirk; Claessens, Frank

    2014-07-01

    Androgens have well-established anabolic actions on skeletal muscle, although the direct effects of the androgen receptor (AR) in muscle remain unclear. We generated satellite cell-specific AR-knockout (satARKO) mice in which the AR is selectively ablated in satellite cells, the muscle precursor cells. Total-limb maximal grip strength is decreased by 7% in satARKO mice, with soleus muscles containing ∼10% more type I fibers and 10% less type IIa fibers than the corresponding control littermates. The weight of the perineal levator ani muscle is markedly reduced (-52%). Thus, muscle AR is involved in fiber-type distribution and force production of the limb muscles, while it is a major determinant of the perineal muscle mass. Surprisingly, myostatin (Mstn), a strong inhibitor of skeletal muscle growth, is one of the most androgen-responsive genes (6-fold reduction in satARKO) through direct transcription activation by the AR. Consequently, muscle hypertrophy in response to androgens is augmented in Mstn-knockout mice. Our finding that androgens induce Mstn signaling to restrain their own anabolic actions has implications for the treatment of muscle wasting disorders.-Dubois, V., Laurent, M. R., Sinnesael, M., Cielen, N., Helsen, C., Clinckemalie, L., Spans, L., Gayan-Ramirez, G., Deldicque, L., Hespel, P., Carmeliet, G., Vanderschueren, D., and Claessens, F. A satellite cell-specific knockout of the androgen receptor reveals myostatin as a direct androgen target in skeletal muscle.

  12. Synaptic abnormalities and cytoplasmic glutamate receptor aggregates in contactin associated protein-like 2/Caspr2 knockout neurons

    PubMed Central

    Varea, Olga; Martin-de-Saavedra, Maria Dolores; Kopeikina, Katherine J.; Schürmann, Britta; Fleming, Hunter J.; Fawcett-Patel, Jessica M.; Bach, Anthony; Jang, Seil; Peles, Elior; Kim, Eunjoon; Penzes, Peter

    2015-01-01

    Central glutamatergic synapses and the molecular pathways that control them are emerging as common substrates in the pathogenesis of mental disorders. Genetic variation in the contactin associated protein-like 2 (CNTNAP2) gene, including copy number variations, exon deletions, truncations, single nucleotide variants, and polymorphisms have been associated with intellectual disability, epilepsy, schizophrenia, language disorders, and autism. CNTNAP2, encoded by Cntnap2, is required for dendritic spine development and its absence causes disease-related phenotypes in mice. However, the mechanisms whereby CNTNAP2 regulates glutamatergic synapses are not known, and cellular phenotypes have not been investigated in Cntnap2 knockout neurons. Here we show that CNTNAP2 is present in dendritic spines, as well as axons and soma. Structured illumination superresolution microscopy reveals closer proximity to excitatory, rather than inhibitory synaptic markers. CNTNAP2 does not promote the formation of synapses and cultured neurons from Cntnap2 knockout mice do not show early defects in axon and dendrite outgrowth, suggesting that CNTNAP2 is not required at this stage. However, mature neurons from knockout mice show reduced spine density and levels of GluA1 subunits of AMPA receptors in spines. Unexpectedly, knockout neurons show large cytoplasmic aggregates of GluA1. Here we characterize, for the first time to our knowledge, synaptic phenotypes in Cntnap2 knockout neurons and reveal a novel role for CNTNAP2 in GluA1 trafficking. Taken together, our findings provide insight into the biological roles of CNTNAP2 and into the pathogenesis of CNTNAP2-associated neuropsychiatric disorders. PMID:25918374

  13. 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.

  14. Increased gabaergic input to ventral tegmental area dopaminergic neurons associated with decreased cocaine reinforcement in mu-opioid receptor knockout mice.

    PubMed

    Mathon, D S; Lesscher, H M B; Gerrits, M A F M; Kamal, A; Pintar, J E; Schuller, A G P; Spruijt, B M; Burbach, J P H; Smidt, M P; van Ree, J M; Ramakers, G M J

    2005-01-01

    There is general agreement that dopaminergic neurons projecting from the ventral tegmental area (VTA) to the nucleus accumbens and prefrontal cortex play a key role in drug reinforcement. The activity of these neurons is strongly modulated by the inhibitory and excitatory input they receive. Activation of mu-opioid receptors, located on GABAergic neurons in the VTA, causes hyperpolarization of these GABAergic neurons, thereby causing a disinhibition of VTA dopaminergic neurons. This effect of mu-opioid receptors upon GABA neurotransmission is a likely mechanism for mu-opioid receptor modulation of drug reinforcement. We studied mu-opioid receptor signaling in relation to cocaine reinforcement in wild-type and mu-opioid receptor knockout mice using a cocaine self-administration paradigm and in vitro electrophysiology. Cocaine self-administration was reduced in mu-opioid receptor knockout mice, suggesting a critical role of mu-opioid receptors in cocaine reinforcement. The frequency of spontaneous inhibitory post-synaptic currents onto dopaminergic neurons in the ventral tegmental area was increased in mu-opioid receptor knockout mice compared with wild-type controls, while the frequency of spontaneous excitatory post-synaptic currents was unaltered. The reduced cocaine self-administration and increased GABAergic input to VTA dopaminergic neurons in mu-opioid receptor knockout mice supports the notion that suppression of GABAergic input onto dopaminergic neurons in the VTA contributes to mu-opioid receptor modulation of cocaine reinforcement. PMID:15664692

  15. 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.

  16. [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

  17. HSL-knockout mouse testis exhibits class B scavenger receptor upregulation and disrupted lipid raft microdomains[S

    PubMed Central

    Casado, María Emilia; Huerta, Lydia; Ortiz, Ana Isabel; Pérez-Crespo, Mirian; Gutiérrez-Adán, Alfonso; Kraemer, Fredric B.; Lasunción, Miguel Ángel; Busto, Rebeca; Martín-Hidalgo, Antonia

    2012-01-01

    There is a tight relationship between fertility and changes in cholesterol metabolism during spermatogenesis. In the testis, class B scavenger receptors (SR-B) SR-BI, SR-BII, and LIMP II mediate the selective uptake of cholesterol esters from HDL, which are hydrolyzed to unesterified cholesterol by hormone-sensitive lipase (HSL). HSL is critical because HSL knockout (KO) male mice are sterile. The aim of the present work was to determine the effects of the lack of HSL in testis on the expression of SR-B, lipid raft composition, and related cell signaling pathways. HSL-KO mouse testis presented altered spermatogenesis associated with decreased sperm counts, sperm motility, and infertility. In wild-type (WT) testis, HSL is expressed in elongated spermatids; SR-BI, in Leydig cells and spermatids; SR-BII, in spermatocytes and spermatids but not in Leydig cells; and LIMP II, in Sertoli and Leydig cells. HSL knockout male mice have increased expression of class B scavenger receptors, disrupted caveolin-1 localization in lipid raft plasma membrane microdomains, and activated phospho-ERK, phospho-AKT, and phospho-SRC in the testis, suggesting that class B scavenger receptors are involved in cholesterol ester uptake for steroidogenesis and spermatogenesis in the testis. PMID:22988039

  18. Paired immunoglobulin-like receptor B knockout does not enhance axonal regeneration or locomotor recovery after spinal cord injury.

    PubMed

    Nakamura, Yuka; Fujita, Yuki; Ueno, Masaki; Takai, Toshiyuki; Yamashita, Toshihide

    2011-01-21

    Myelin components that inhibit axonal regeneration are believed to contribute significantly to the lack of axonal regeneration noted in the adult central nervous system. Three proteins found in myelin, Nogo, myelin-associated glycoprotein, and oligodendrocyte-myelin glycoprotein, inhibit neurite outgrowth in vitro. All of these proteins interact with the same receptors, namely, the Nogo receptor (NgR) and paired immunoglobulin-like receptor B (PIR-B). As per previous reports, corticospinal tract (CST) regeneration is not enhanced in NgR-knock-out mice after spinal cord injury. Therefore, we assessed CST regeneration in PIR-B-knock-out mice. We found that hindlimb motor function, as assessed using the Basso mouse scale, footprint test, inclined plane test, and beam walking test, did not differ between the PIR-B-knock-out and wild-type mice after dorsal hemisection of the spinal cord. Further, tracing of the CST fibers after injury did not reveal enhanced axonal regeneration or sprouting in the CST of the PIR-B-knock-out mice. Systemic administration of NEP1-40, a NgR antagonist, to PIR-B knock-out mice did not enhance the regenerative response. These results indicate that PIR-B knock-out is not sufficient to induce extensive axonal regeneration after spinal cord injury. PMID:21087927

  19. Data on Arc and Zif268 expression in the brain of the α-2A adrenergic receptor knockout mouse.

    PubMed

    Sanders, Jeff

    2016-06-01

    The α2-adrenergic receptor (α2-AR) is widely distributed in the brain with distinct roles for α2-AR subtypes (A, B and C). In this article, data are provided on Activity Regulated Cytoskeleton Associated Protein (Arc) and Zif268 expression in the brain of the α2A-AR knockout (α2A-AR KO) mouse. These data are supplemental to an original research article examining Arc and Zif268 expression in rats injected with the α2-AR antagonist, RX821002 (http://dx.doi.org/10.1016/j.neulet.2015.12.002. [1]). PMID:26952134

  20. Characterization of the retina in the alpha7 nicotinic acetylcholine receptor knockout mouse

    NASA Astrophysics Data System (ADS)

    Smith, Marci L.

    Acetylcholine receptors (AChRs) are involved in visual processing and are expressed by inner retinal neurons in all species studied to date (Keyser et al., 2000; Dmitrieva et al., 2007; Liu et al., 2009), but their distribution in the mouse retina remains unknown. Reductions in alpha7 nicotinic AChRs (nAChRs) are thought to contribute to memory and visual deficits observed in Alzheimer's and schizophrenia (Coyle et al., 1983; Nordberg et al., 1999; Leonard et al., 2006). However, the alpha7 nAChR knockout (KO) mouse has a mild phenotype (Paylor et al., 1998; Fernandes et al., 2006; Young et al., 2007; Origlia et al., 2012). The purpose of this study was to determine the expression of AChRs in wildtype (WT) mouse retina and to assess whether up-regulation of other AChRs in the alpha7 nAChR KO retina may explain the minimal deficits described in the KO mouse. Reverse-transcriptase PCR (RT-PCR) showed that mRNA transcripts for alpha2-7, alpha 9, alpha10, beta2-4 nAChR subunits and m1-m5 muscarinic AChR (mAChR) subtypes were present in WT murine retina. Western blot analysis confirmed the presence of alpha3-5, alpha9, and m1-m5 AChR proteins and immunohistochemical analysis demonstrated nAChR and mAChR proteins expressed by subsets of bipolar, amacrine and ganglion cells. This is the first reported expression of alpha9 and alpha10 nAChR transcripts and alpha9 nAChR proteins in the retina of any species. Quantitative RT-PCR (qPCR) showed changes in AChR transcript expression in the alpha7 nAChR KO mouse retina relative to WT. Within whole retina alpha2, alpha9, alpha10, beta4, m1 and m4 AChR transcripts were up-regulated, while alpha5 nAChR transcripts were down-regulated. However, cell populations showed subtle differences; m4 mAChR transcripts were up-regulated in the ganglion cell layer and outer portion of the inner nuclear layer (oINL),while beta4 nAChR transcript up-regulation was limited to the oINL. Surprisingly, alpha2, alpha9, beta4, m2 and m4 transcripts were

  1. 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.

  2. 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

  3. Genetic background can result in a marked or minimal effect of gene knockout (GPR55 and CB2 receptor) in experimental autoimmune encephalomyelitis models of multiple sclerosis.

    PubMed

    Sisay, Sofia; Pryce, Gareth; Jackson, Samuel J; Tanner, Carolyn; Ross, Ruth A; Michael, Gregory J; Selwood, David L; Giovannoni, Gavin; Baker, David

    2013-01-01

    Endocannabinoids and some phytocannabinoids bind to CB1 and CB2 cannabinoid receptors, transient receptor potential vanilloid one (TRPV1) receptor and the orphan G protein receptor fifty-five (GPR55). Studies using C57BL/10 and C57BL/6 (Cnr2 (tm1Zim)) CB2 cannabinoid receptor knockout mice have demonstrated an immune-augmenting effect in experimental autoimmune encephalomyelitis (EAE) models of multiple sclerosis. However, other EAE studies in Biozzi ABH mice often failed to show any treatment effect of either CB2 receptor agonism or antagonism on inhibition of T cell autoimmunity. The influence of genetic background on the induction of EAE in endocannabinoid system-related gene knockout mice was examined. It was found that C57BL/6.GPR55 knockout mice developed less severe disease, notably in female mice, following active induction with myelin oligodendrocyte glycoprotein 35-55 peptide. In contrast C57BL/6.CB2 (Cnr2 (Dgen)) receptor knockout mice developed augmented severity of disease consistent with the genetically and pharmacologically-distinct, Cnr2 (tm1Zim) mice. However, when the knockout gene was bred into the ABH mouse background and EAE induced with spinal cord autoantigens the immune-enhancing effect of CB2 receptor deletion was lost. Likewise CB1 receptor and transient receptor potential vanilloid one knockout mice on the ABH background demonstrated no alteration in immune-susceptibility, in terms of disease incidence and severity of EAE, in contrast to that reported in some C57BL/6 mouse studies. Furthermore the immune-modulating influence of GPR55 was marginal on the ABH mouse background. Whilst sedative doses of tetrahydrocannabinol could induce immunosuppression, this was associated with a CB1 receptor rather than a CB2 receptor-mediated effect. These data support the fact that non-psychoactive doses of medicinal cannabis have a marginal influence on the immune response in MS. Importantly, it adds a note of caution for the translational value of some

  4. Genetic Background Can Result in a Marked or Minimal Effect of Gene Knockout (GPR55 and CB2 Receptor) in Experimental Autoimmune Encephalomyelitis Models of Multiple Sclerosis

    PubMed Central

    Jackson, Samuel J.; Tanner, Carolyn; Ross, Ruth A.; Michael, Gregory J.; Selwood, David L.; Giovannoni, Gavin; Baker, David

    2013-01-01

    Endocannabinoids and some phytocannabinoids bind to CB1 and CB2 cannabinoid receptors, transient receptor potential vanilloid one (TRPV1) receptor and the orphan G protein receptor fifty-five (GPR55). Studies using C57BL/10 and C57BL/6 (Cnr2tm1Zim) CB2 cannabinoid receptor knockout mice have demonstrated an immune-augmenting effect in experimental autoimmune encephalomyelitis (EAE) models of multiple sclerosis. However, other EAE studies in Biozzi ABH mice often failed to show any treatment effect of either CB2 receptor agonism or antagonism on inhibition of T cell autoimmunity. The influence of genetic background on the induction of EAE in endocannabinoid system-related gene knockout mice was examined. It was found that C57BL/6.GPR55 knockout mice developed less severe disease, notably in female mice, following active induction with myelin oligodendrocyte glycoprotein 35-55 peptide. In contrast C57BL/6.CB2 (Cnr2Dgen) receptor knockout mice developed augmented severity of disease consistent with the genetically and pharmacologically-distinct, Cnr2tm1Zim mice. However, when the knockout gene was bred into the ABH mouse background and EAE induced with spinal cord autoantigens the immune-enhancing effect of CB2 receptor deletion was lost. Likewise CB1 receptor and transient receptor potential vanilloid one knockout mice on the ABH background demonstrated no alteration in immune-susceptibility, in terms of disease incidence and severity of EAE, in contrast to that reported in some C57BL/6 mouse studies. Furthermore the immune-modulating influence of GPR55 was marginal on the ABH mouse background. Whilst sedative doses of tetrahydrocannabinol could induce immunosuppression, this was associated with a CB1 receptor rather than a CB2 receptor-mediated effect. These data support the fact that non-psychoactive doses of medicinal cannabis have a marginal influence on the immune response in MS. Importantly, it adds a note of caution for the translational value of some

  5. A muscle-specific knockout implicates nuclear receptor coactivator MED1 in the regulation of glucose and energy metabolism.

    PubMed

    Chen, Wei; Zhang, Xiaoting; Birsoy, Kivanc; Roeder, Robert G

    2010-06-01

    As conventional transcriptional factors that are activated in diverse signaling pathways, nuclear receptors play important roles in many physiological processes that include energy homeostasis. The MED1 subunit of the Mediator coactivator complex plays a broad role in nuclear receptor-mediated transcription by anchoring the Mediator complex to diverse promoter-bound nuclear receptors. Given the significant role of skeletal muscle, in part through the action of nuclear receptors, in glucose and fatty acid metabolism, we generated skeletal muscle-specific Med1 knockout mice. Importantly, these mice show enhanced insulin sensitivity and improved glucose tolerance as well as resistance to high-fat diet-induced obesity. Furthermore, the white muscle of these mice exhibits increased mitochondrial density and expression of genes specific to type I and type IIA fibers, indicating a fast-to-slow fiber switch, as well as markedly increased expression of the brown adipose tissue-specific UCP-1 and Cidea genes that are involved in respiratory uncoupling. These dramatic results implicate MED1 as a powerful suppressor in skeletal muscle of genetic programs implicated in energy expenditure and raise the significant possibility of therapeutical approaches for metabolic syndromes and muscle diseases through modulation of MED1-nuclear receptor interactions.

  6. Increased brain monoaminergic tone after the NMDA receptor GluN2A subunit gene knockout is responsible for resistance to the hypnotic effect of nitrous oxide.

    PubMed

    Petrenko, Andrey B; Yamakura, Tomohiro; Kohno, Tatsuro; Sakimura, Kenji; Baba, Hiroshi

    2013-01-01

    N-methyl-d-aspartate (NMDA) receptors can be inhibited by inhalational anesthetics in vitro at clinically relevant concentrations. Here, to clarify the role of NMDA receptors in anesthetic-induced unconsciousness, we examined the hypnotic properties of isoflurane, sevoflurane and nitrous oxide in NMDA receptor GluN2A subunit knockout mice. The hypnotic properties of inhalational anesthetics were evaluated in mice in the loss of righting reflex (LORR) assay by measuring the 50% concentration for LORR (LORR ED(50)). Knockout mice displayed isoflurane and sevoflurane LORR ED(50) values similar to wild-type controls, indicating no significant contribution of these receptors to the hypnotic action of halogenated anesthetics. However, compared with wild-type controls, mutant mice displayed larger isoflurane LORR ED(50) values in the presence of nitrous oxide, indicating a resistance to this gaseous anesthetic. Knockout mice have enhanced brain monoaminergic activity which occurs secondary to NMDA receptor dysfunction, and the observed resistance to the isoflurane LORR ED(50)-sparing effect of nitrous oxide could be abolished by pretreatment with the dopamine D(2) receptor antagonist droperidol or with the serotonin 5-HT(2A) receptor antagonist ketanserin. Thus, resistance to nitrous oxide in knockout mice appears to be a secondary phenomenon of monoaminergic origin and not a direct result of impaired NMDA receptor function. Our results indicate that NMDA receptors are not critically involved in the hypnotic action of conventionally-used inhalational anesthetics. Also, they suggest that increased brain monoaminergic tone can diminish the effects of general anesthesia. Finally, they provide further evidence that changes secondary to genetic manipulation can explain the results obtained in global knockouts. PMID:23123346

  7. 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.

  8. 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.

  9. Electrical interfacing of neurotransmitter receptor and field effect transistor

    NASA Astrophysics Data System (ADS)

    Peitz, I.; Fromherz, P.

    2009-10-01

    The interfacing of a ligand-gated ion channel to a transistor is studied. It relies on the transduction of ion current to a voltage in a cell-transistor junction. For the first time, a genetically modified cell is used without external driving voltage as applied by a patch-pipette. Using a core-coat conductor model, we show that an autonomous dynamics gives rise to a signal if a driving voltage is provided by potassium channels, and if current compensation is avoided by an inhomogeneous activation of channels. In a proof-of-principle experiment, we transfect HEK293 cells with the serotonin receptor 5-HT3A and the potassium channel Kv1.3. The interfacing is characterized under voltage-clamp with a negative transistor signal for activated 5-HT3A and a positive signal for activated Kv1.3. Without patch-pipette, a biphasic transient is induced by serotonin. The positive wave is assigned to 5-HT3A receptors in the free membrane that drive a potassium outward current through the adherent membrane. The negative wave is attributed to 5-HT3A receptors in the adherent membrane that are activated with a delay due to serotonin diffusion. The implementation of a receptor-cell-transistor device is a fundamental step in the development of biosensors that combine high specificity and universal microelectronic readout.

  10. Interaction between 5-HT(1A) and 5-HT(1B) receptors: effects of 8-OH-DPAT-induced hypothermia in 5-HT(1B) receptor knockout mice.

    PubMed

    Gardier, A M; Gruwez, B; Trillat, A C; Jacquot, C; Hen, R; Bourin, M

    2001-06-15

    To test for adaptive compensatory changes that may have occurred in the functional activity of somatodendritic 5-HT(1A) receptors during the development of constitutive "knockout" mice lacking the 5-HT(1B) receptor subtype (5-HT(1B) -/- KO), we assayed for decrease in body temperature induced by an acute subcutaneous injection of the 5-HT(1A) receptor agonist, 8-hydroxy 2(di-n-propyl(amino)tetralin (8-OH-DPAT), either alone or in the presence of a selective 5-HT(1A) receptor antagonist, N-[4-(2-methoxyphenyl)-1-piperazinyl]-N-(2-pyridinyl) cyclo-hexanecarboxamide (WAY 100635). We compared dose-response curves, time course study, calculated ED(50) values (potency), maximal response to 8-OH-DPAT (efficacy) as well as measurements of the dose-dependent blockade of this response by WAY 100635 between wild-type controls and mutant mice. We found a higher efficacy of 8-OH-DPAT-induced hypothermia in 5-HT(1B) -/- KO compared to wild-type mice suggesting that an adaptive thermoregulatory process involving the functional activity of somatodendritic 5-HT(1A) receptors is altered in mutant mice lacking 5-HT(1B) receptors.

  11. 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.

  12. Toll-like receptor 4 knockout alleviates paraquat-induced cardiomyocyte contractile dysfunction through an autophagy-dependent mechanism.

    PubMed

    Wang, Shuyi; Zhu, Xiaoling; Xiong, Lize; Zhang, Yingmei; Ren, Jun

    2016-08-22

    Paraquat, a quarternary nitrogen herbicide, is a toxic prooxidant leading to multi-organ failure including the heart although the underlying mechanism remains poorly understood. This study was designed to examine the role of the innate proinflammatory mediator toll-like receptor 4 (TLR4) in paraquat-induced cardiac contractile anomalies and the underlying mechanisms involved with a focus on autophagy, a conservative machinery governing protein and organelle degradation and recycling for cardiac homeostasis. Wild-type (WT) and TLR4 knockout (TLR4(-/-)) mice were challenged with paraquat (45mg/kg, i.p.) for 48h. Paraquat challenge did not affect mRNA levels of TLR2, TLR4 and TLR9 in WT mice nor did paraquat treatment alter TREM-1 levels. Paraquat challenge elicited cardiac mechanical defects including compromised cardiomyocyte contractile function, intracellular Ca(2+) handling, and overt autophagy as manifested by increased LC3BII-to-LC3BI ratio, Atg5, Atg7 and p62 levels. Interestingly, TLR4 knockout significantly attenuated paraquat-induced cardiac contractile and intracellular Ca(2+) derangement as well as alterations of autophagy markers. Paraquat-elicited changes in cardiac autophagy markers (LC3BII, LC3BII-to-LC3BI ratio and p62) were augmented by lysosomal inhibition using bafilomycin A1 in WT mice. TLR4 knockout significantly attenuated or negated paraquat-elicited increase in LC3BII, LC3BII-to-LC3BI ratio and p62 levels in the presence of lysosomal inhibition. In addition, paraquat challenge promoted phosphorylation of AMPK while suppressing the phosphorylation of mTOR and ULK1 (the autophagy inhibitory Ser(757)), the effects of which were significantly attenuated by TLR4 ablation. In vitro study revealed that AMPK activation using AICAR or mTOR inhibition using rapamycin effectively negated the beneficial cardiomyocyte mechanical effects of TLR4 inhibition (CLI-095) against paraquat toxicity, supporting a permissive role for AMPK-mTOR in TLR4 inhibition

  13. Toll-like receptor 4 knockout alleviates paraquat-induced cardiomyocyte contractile dysfunction through an autophagy-dependent mechanism.

    PubMed

    Wang, Shuyi; Zhu, Xiaoling; Xiong, Lize; Zhang, Yingmei; Ren, Jun

    2016-08-22

    Paraquat, a quarternary nitrogen herbicide, is a toxic prooxidant leading to multi-organ failure including the heart although the underlying mechanism remains poorly understood. This study was designed to examine the role of the innate proinflammatory mediator toll-like receptor 4 (TLR4) in paraquat-induced cardiac contractile anomalies and the underlying mechanisms involved with a focus on autophagy, a conservative machinery governing protein and organelle degradation and recycling for cardiac homeostasis. Wild-type (WT) and TLR4 knockout (TLR4(-/-)) mice were challenged with paraquat (45mg/kg, i.p.) for 48h. Paraquat challenge did not affect mRNA levels of TLR2, TLR4 and TLR9 in WT mice nor did paraquat treatment alter TREM-1 levels. Paraquat challenge elicited cardiac mechanical defects including compromised cardiomyocyte contractile function, intracellular Ca(2+) handling, and overt autophagy as manifested by increased LC3BII-to-LC3BI ratio, Atg5, Atg7 and p62 levels. Interestingly, TLR4 knockout significantly attenuated paraquat-induced cardiac contractile and intracellular Ca(2+) derangement as well as alterations of autophagy markers. Paraquat-elicited changes in cardiac autophagy markers (LC3BII, LC3BII-to-LC3BI ratio and p62) were augmented by lysosomal inhibition using bafilomycin A1 in WT mice. TLR4 knockout significantly attenuated or negated paraquat-elicited increase in LC3BII, LC3BII-to-LC3BI ratio and p62 levels in the presence of lysosomal inhibition. In addition, paraquat challenge promoted phosphorylation of AMPK while suppressing the phosphorylation of mTOR and ULK1 (the autophagy inhibitory Ser(757)), the effects of which were significantly attenuated by TLR4 ablation. In vitro study revealed that AMPK activation using AICAR or mTOR inhibition using rapamycin effectively negated the beneficial cardiomyocyte mechanical effects of TLR4 inhibition (CLI-095) against paraquat toxicity, supporting a permissive role for AMPK-mTOR in TLR4 inhibition

  14. Differential actions of orexin receptors in brainstem cholinergic and monoaminergic neurons revealed by receptor knockouts: implications for orexinergic signaling in arousal and narcolepsy

    PubMed Central

    Kohlmeier, Kristi A.; Tyler, Christopher J.; Kalogiannis, Mike; Ishibashi, Masaru; Kristensen, Morten P.; Gumenchuk, Iryna; Chemelli, Richard M.; Kisanuki, Yaz Y.; Yanagisawa, Masashi; Leonard, Christopher S.

    2013-01-01

    Orexin neuropeptides influence multiple homeostatic functions and play an essential role in the expression of normal sleep-wake behavior. While their two known receptors (OX1 and OX2) are targets for novel pharmacotherapeutics, the actions mediated by each receptor remain largely unexplored. Using brain slices from mice constitutively lacking either receptor, we used whole-cell and Ca2+ imaging methods to delineate the cellular actions of each receptor within cholinergic [laterodorsal tegmental nucleus (LDT)] and monoaminergic [dorsal raphe (DR) and locus coeruleus (LC)] brainstem nuclei—where orexins promote arousal and suppress REM sleep. In slices from OX−/−2 mice, orexin-A (300 nM) elicited wild-type responses in LDT, DR, and LC neurons consisting of a depolarizing current and augmented voltage-dependent Ca2+ transients. In slices from OX−/−1 mice, the depolarizing current was absent in LDT and LC neurons and was attenuated in DR neurons, although Ca2+-transients were still augmented. Since orexin-A produced neither of these actions in slices lacking both receptors, our findings suggest that orexin-mediated depolarization is mediated by both receptors in DR, but is exclusively mediated by OX1 in LDT and LC neurons, even though OX2 is present and OX2 mRNA appears elevated in brainstems from OX−/−1 mice. Considering published behavioral data, these findings support a model in which orexin-mediated excitation of mesopontine cholinergic and monoaminergic neurons contributes little to stabilizing spontaneous waking and sleep bouts, but functions in context-dependent arousal and helps restrict muscle atonia to REM sleep. The augmented Ca2+ transients produced by both receptors appeared mediated by influx via L-type Ca2+ channels, which is often linked to transcriptional signaling. This could provide an adaptive signal to compensate for receptor loss or prolonged antagonism and may contribute to the reduced severity of narcolepsy in single receptor

  15. Glutamate receptor mGlu2 and mGlu3 knockout striata are dopamine supersensitive, with elevated D2(High) receptors and marked supersensitivity to the dopamine agonist (+)PHNO.

    PubMed

    Seeman, Philip; Battaglia, Giuseppe; Corti, Corrado; Corsi, Mauro; Bruno, Valeria

    2009-03-01

    The finding that the mGlu2/3 metabotropic glutamate receptor agonist, LY404039, improves clinical symptoms in schizophrenia warrants a search for a possible interaction between mGlu2/3 receptors and dopamine D2 receptors. Here, this topic is examined in striatal tissue of mice lacking either mGlu2 or mGlu3 receptor. Such mice are known to be behaviorally supersensitive to dopamine receptor agonists. Therefore, to determine the basis of this dopamine supersensitivity, the proportion of dopamine D2(High) receptors was measured in the striata of mGlu2 and mGlu3 receptor knockout mice. The proportion of D2(High) receptors was found to be elevated by 220% in the striata of both knockouts. To measure the functional dopamine supersensitivity, the D2 agonist (+)PHNO was used to stimulate the incorporation of GTP-gamma-S in the striatal homogenates in the presence of drugs that blocked the dopamine D1, D3, and D5 receptors. Compared with control striata, the mGlu2 receptor knockout tissues were 67-fold more sensitive to (+)PHNO, while the mGlu3 receptor knockout tissues were 17-fold more sensitive. These data suggest that group II mGlu receptors-mGlu2 receptors in particular-may normally regulate D2 receptors by reducing the proportion of high-affinity D2 receptors in membranes. Such regulation may contribute to the antipsychotic action of mGlu2/3 receptor agonists. PMID:19084908

  16. 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

  17. Enhanced histaminergic neurotransmission and sleep-wake alterations, a study in histamine H3-receptor knock-out mice.

    PubMed

    Gondard, Elise; Anaclet, Christelle; Akaoka, Hidéo; Guo, Rui-Xian; Zhang, Mei; Buda, Colette; Franco, Patricia; Kotani, Hidehito; Lin, Jian-Sheng

    2013-05-01

    Long-term abolition of a brain arousal system impairs wakefulness (W), but little is known about the consequences of long-term enhancement. The brain histaminergic arousal system is under the negative control of H3-autoreceptors whose deletion results in permanent enhancement of histamine (HA) turnover. In order to determine the consequences of enhancement of the histaminergic system, we compared the cortical EEG and sleep-wake states of H3-receptor knockout (H3R-/-) and wild-type mouse littermates. We found that H3R-/-mice had rich phenotypes. On the one hand, they showed clear signs of enhanced HA neurotransmission and vigilance, i.e., a higher EEG θ power during spontaneous W and a greater extent of W or sleep restriction during behavioral tasks, including environmental change, locomotion, and motivation tests. On the other hand, during the baseline dark period, they displayed deficient W and signs of sleep deterioration, such as pronounced sleep fragmentation and reduced cortical slow activity during slow wave sleep (SWS), most likely due to a desensitization of postsynaptic histaminergic receptors as a result of constant HA release. Ciproxifan (H3-receptor inverse agonist) enhanced W in wild-type mice, but not in H3R-/-mice, indicating a functional deletion of H3-receptors, whereas triprolidine (postsynaptic H1-receptor antagonist) or α-fluoromethylhistidine (HA-synthesis inhibitor) caused a greater SWS increase in H3R-/- than in wild-type mice, consistent with enhanced HA neurotransmission. These sleep-wake characteristics and the obesity phenotypes previously reported in this animal model suggest that chronic enhancement of histaminergic neurotransmission eventually compromises the arousal system, leading to sleep-wake, behavioral, and metabolic disorders similar to those caused by voluntary sleep restriction in humans.

  18. Normal sleep homeostasis and lack of epilepsy phenotype in GABA A receptor alpha3 subunit-knockout mice.

    PubMed

    Winsky-Sommerer, R; Knapman, A; Fedele, D E; Schofield, C M; Vyazovskiy, V V; Rudolph, U; Huguenard, J R; Fritschy, J-M; Tobler, I

    2008-06-23

    Thalamo-cortical networks generate specific patterns of oscillations during distinct vigilance states and epilepsy, well characterized by electroencephalography (EEG). Oscillations depend on recurrent synaptic loops, which are controlled by GABAergic transmission. In particular, GABA A receptors containing the alpha3 subunit are expressed predominantly in cortical layer VI and thalamic reticular nucleus (nRT) and regulate the activity and firing pattern of neurons in relay nuclei. Therefore, ablation of these receptors by gene targeting might profoundly affect thalamo-cortical oscillations. Here, we investigated the role of alpha3-GABA A receptors in regulating vigilance states and seizure activity by analyzing chronic EEG recordings in alpha3 subunit-knockout (alpha3-KO) mice. The presence of postsynaptic alpha3-GABA A receptors/gephyrin clusters in the nRT and GABA A-mediated synaptic currents in acute thalamic slices was also examined. EEG spectral analysis showed no difference between genotypes during non rapid-eye movement (NREM) sleep or at waking-NREM sleep transitions. EEG power in the spindle frequency range (10-15 Hz) was significantly lower at NREM-REM sleep transitions in mutant compared with wild-type mice. Enhancement of sleep pressure by 6 h sleep deprivation did not reveal any differences in the regulation of EEG activities between genotypes. Finally, the waking EEG showed a slightly larger power in the 11-13-Hz band in alpha3-KO mice. However, neither behavior nor the waking EEG showed alterations suggestive of absence seizures. Furthermore, alpha3-KO mice did not differ in seizure susceptibility in a model of temporal lobe epilepsy. Strikingly, despite the disruption of postsynaptic gephyrin clusters, whole-cell patch clamp recordings revealed intact inhibitory synaptic transmission in the nRT of alpha3-KO mice. These findings show that the lack of alpha3-GABA(A) receptors is extensively compensated for to preserve the integrity of thalamo

  19. Enhanced Histaminergic Neurotransmission and Sleep-Wake Alterations, a Study in Histamine H3-Receptor Knock-Out Mice

    PubMed Central

    Gondard, Elise; Anaclet, Christelle; Akaoka, Hidéo; Guo, Rui-Xian; Zhang, Mei; Buda, Colette; Franco, Patricia; Kotani, Hidehito; Lin, Jian-Sheng

    2013-01-01

    Long-term abolition of a brain arousal system impairs wakefulness (W), but little is known about the consequences of long-term enhancement. The brain histaminergic arousal system is under the negative control of H3-autoreceptors whose deletion results in permanent enhancement of histamine (HA) turnover. In order to determine the consequences of enhancement of the histaminergic system, we compared the cortical EEG and sleep-wake states of H3-receptor knockout (H3R−/−) and wild-type mouse littermates. We found that H3R−/−mice had rich phenotypes. On the one hand, they showed clear signs of enhanced HA neurotransmission and vigilance, i.e., a higher EEG θ power during spontaneous W and a greater extent of W or sleep restriction during behavioral tasks, including environmental change, locomotion, and motivation tests. On the other hand, during the baseline dark period, they displayed deficient W and signs of sleep deterioration, such as pronounced sleep fragmentation and reduced cortical slow activity during slow wave sleep (SWS), most likely due to a desensitization of postsynaptic histaminergic receptors as a result of constant HA release. Ciproxifan (H3-receptor inverse agonist) enhanced W in wild-type mice, but not in H3R−/−mice, indicating a functional deletion of H3-receptors, whereas triprolidine (postsynaptic H1-receptor antagonist) or α-fluoromethylhistidine (HA-synthesis inhibitor) caused a greater SWS increase in H3R−/− than in wild-type mice, consistent with enhanced HA neurotransmission. These sleep-wake characteristics and the obesity phenotypes previously reported in this animal model suggest that chronic enhancement of histaminergic neurotransmission eventually compromises the arousal system, leading to sleep-wake, behavioral, and metabolic disorders similar to those caused by voluntary sleep restriction in humans. PMID:23303066

  20. Assessment of ethanol consumption and water drinking by NPY Y(2) receptor knockout mice.

    PubMed

    Thiele, Todd E; Naveilhan, Philippe; Ernfors, Patrik

    2004-06-01

    In recent years, pharmacological and genetic evidence have emerged suggesting that neuropeptide Y (NPY) and the NPY Y(1) receptor are involved with neurobiological responses to ethanol. Pharmacological data implicate a role for the NPY Y(2) receptor in ethanol self-administration. The purpose of the present study was to determine if genetic mutation of the Y(2) receptor would modulate ethanol consumption and/or ethanol-induced sedation. Here, we report that mutant mice lacking the NPY Y(2) receptor (Y(2)(-/-)), when maintained on a mixed 50% 129/ SvJ x 50 % Balb/cJ background, drink significantly less of solutions containing 3 or 6% (v/v) ethanol relative to wild-type (Y(2)(+/+)) mice. These mice drink normal amounts of solutions containing sucrose or quinine, have normal blood ethanol clearance, and show normal sensitivity to ethanol-induced sedation. However, Y(2)(-/-) mice that are backcrossed to a Balb/cJ background show normal consumption of ethanol, indicating that the contributions of the NPY Y(2) receptor to ethanol consumption are genetic background dependent. Consistent with previous data suggesting that NPY modulates water drinking, Y(2)(-/-) mice of both genetic backgrounds consume significantly more water than Y(2)(+/+) mice. The present results suggest roles for the NPY Y(2) receptor in the modulation of ethanol and water consumption.

  1. Dysfunctional Presynaptic M2 Receptors in the Presence of Chronically High Acetylcholine Levels: Data from the PRiMA Knockout Mouse

    PubMed Central

    Mohr, Franziska; Krejci, Eric; Zimmermann, Martina; Klein, Jochen

    2015-01-01

    The muscarinic M2 receptor (M2R) acts as a negative feedback regulator in central cholinergic systems. Activation of the M2 receptor limits acetylcholine (ACh) release, especially when ACh levels are increased because acetylcholinesterase (AChE) activity is acutely inhibited. Chronically high ACh levels in the extracellular space, however, were reported to down-regulate M2R to various degrees. In the present study, we used the PRiMA knockout mouse which develops severely reduced AChE activity postnatally to investigate ACh release, and we used microdialysis to investigate whether the function of M2R to reduce ACh release in vivo was impaired in adult PRiMA knockout mice. We first show that striatal and hippocampal ACh levels, while strongly increased, still respond to AChE inhibitors. Infusion or injection of oxotremorine, a muscarinic M2 agonist, reduced ACh levels in wild-type mice but did not significantly affect ACh levels in PRiMA knockout mice or in wild-type mice in which ACh levels were artificially increased by infusion of neostigmine. Scopolamine, a muscarinic antagonist, increased ACh levels in wild-type mice receiving neostigmine, but not in wild-type mice or in PRiMA knockout mice. These results demonstrate that M2R are dysfunctional and do not affect ACh levels in PRiMA knockout mice, likely because of down-regulation and/or loss of receptor-effector coupling. Remarkably, this loss of function does not affect cognitive functions in PRiMA knockout mice. Our results are discussed in the context of AChE inhibitor therapy as used in dementia. PMID:26506622

  2. Specific regions display altered grey matter volume in μ-opioid receptor knockout mice: MRI voxel-based morphometry

    PubMed Central

    Sasaki, Kazumasu; Sumiyoshi, Akira; Nonaka, Hiroi; Kasahara, Yoshiyuki; Ikeda, Kazutaka; Hall, F Scott; Uhl, George R; Watanabe, Masahiko; Kawashima, Ryuta; Sora, Ichiro

    2015-01-01

    BACKGROUND AND PURPOSE μ Opioid receptor knockout (MOP-KO) mice display several behavioural differences from wild-type (WT) littermates including differential responses to nociceptive stimuli. Brain structural changes have been tied to behavioural alterations noted in transgenic mice with targeting of different genes. Hence, we assess the brain structure of MOP-KO mice. EXPERIMENTAL APPROACH Magnetic resonance imaging (MRI) voxel-based morphometry (VBM) and histological methods were used to identify structural differences between extensively backcrossed MOP-KO mice and WT mice. KEY RESULTS MOP-KO mice displayed robust increases in regional grey matter volume in olfactory bulb, several hypothalamic nuclei, periaqueductal grey (PAG) and several cerebellar areas, most confirmed by VBM analysis. The largest increases in grey matter volume were detected in the glomerular layer of the olfactory bulb, arcuate nucleus of hypothalamus, ventrolateral PAG (VLPAG) and cerebellar regions including paramedian and cerebellar lobules. Histological analyses confirm several of these results, with increased VLPAG cell numbers and increased thickness of the olfactory bulb granule cell layer and cerebellar molecular and granular cell layers. CONCLUSIONS AND IMPLICATIONS MOP deletion causes previously undescribed structural changes in specific brain regions, but not in all regions with high MOP receptor densities (e.g. thalamus, nucleus accumbens) or that exhibit adult neurogenesis (e.g. hippocampus). Volume differences in hypothalamus and PAG may reflect behavioural changes including hyperalgesia. Although the precise relationship between volume change and MOP receptor deletion was not determined from this study alone, these findings suggest that levels of MOP receptor expression may influence a broader range of neural structure and function in humans than previously supposed. LINKED ARTICLES This article is part of a themed section on Opioids: New Pathways to Functional Selectivity

  3. Decreased Incentive Motivation Following Knockout or Acute Blockade of the Serotonin Transporter: Role of the 5-HT2C Receptor.

    PubMed

    Browne, Caleb J; Fletcher, Paul J

    2016-09-01

    Acute pharmacological elevation of serotonin (5-hydroxytryptamine; 5-HT) activity decreases operant responding for primary reinforcers, suggesting that 5-HT reduces incentive motivation. The mechanism by which 5-HT alters incentive motivation is unknown, but parallel evidence that 5-HT2C receptor agonists also reduce responding for primary reinforcers implicates this receptor as a potential candidate. These experiments examined whether chronic and acute disruptions of serotonin transporter (SERT) activity altered incentive motivation, and whether the 5-HT2C receptor mediated the effects of elevated 5-HT on behavior. To assess incentive motivation, we measured responding for three different reinforcers: a primary reinforcer (saccharin), a conditioned reinforcer (CRf), and an unconditioned sensory reinforcer (USRf). In the chronic condition, responding was compared between SERT knockout (SERT-KO) mice and their wild-type littermates. In the acute condition, responding was examined in wild-type mice following treatment with 10 or 20 mg/kg citalopram, or its vehicle. The ability of the selective 5-HT2C antagonist SB 242084 to prevent the effects of SERT-KO and citalopram on responding was subsequently examined. Both SERT-KO and citalopram reduced responding for saccharin, a CRf, and a USRf. Treatment with SB 242084 enhanced responding for a CRf and a USRf in SERT-KO mice and blocked the effects of citalopram on CRf and USRf responding. However, SB 242084 was unable to prevent the effects of SERT-KO or citalopram on responding for saccharin. These results support a powerful inhibitory function for 5-HT in the control of incentive motivation, and indicate that the 5-HT2C receptor mediates these effects of 5-HT in a reinforcer-dependent manner. PMID:27125304

  4. Hypoglycemia, hyperglucagonemia, and fetoplacental defects in glucagon receptor knockout mice: a role for glucagon action in pregnancy maintenance.

    PubMed

    Ouhilal, Sophia; Vuguin, Patricia; Cui, Lingguang; Du, Xiu-Quan; Gelling, Richard W; Reznik, Sandra E; Russell, Robert; Parlow, Albert F; Karpovsky, Clara; Santoro, Nanette; Charron, Maureen J

    2012-03-01

    Alterations in insulin signaling as well as insulin action predispose to infertility as well as adverse pregnancy outcomes; however, little is known about the role of glucagon signaling in reproduction. The glucagon receptor knockout (Gcgr(-/-)) mouse created by our laboratory was used to define the role of glucagon signaling in maintaining normal reproduction. In this mouse model, lack of glucagon signaling did not alter the hypothalamic-pituitary-ovarian axis. Pregnant Gcgr(-/-) female mice displayed persistent hypoglycemia and hyperglucagonemia. Gcgr(-/-) pregnancies were associated with decreased fetal weight, increased late-gestation fetal demise, and significant abnormalities of placentation. Gcgr(-/-) placentas contained areas of extensive mineralization, fibrinoid necrosis, narrowing of the vascular channels, and a thickened interstitium associated with trophoblast hyperplasia. Absent glucagon signaling did not alter glycogen content in Gcgr(-/-) placentas but significantly downregulated genes that control growth, adrenergic signaling, vascularization, oxidative stress, and G protein-coupled receptors. Our data suggest that, similarly to insulin, glucagon action contributes to normal female reproductive function.

  5. 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.

  6. Development of Accelerated Coronary Atherosclerosis Model Using Low Density Lipoprotein Receptor Knock-Out Swine with Balloon Injury

    PubMed Central

    Ogita, Manabu; Miyauchi, Katsumi; Onishi, Akira; Tsuboi, Shuta; Wada, Hideki; Konishi, Hirokazu; Naito, Ryo; Dohi, Tomotaka; Kasai, Takatoshi; Kojima, Yuko; Schwartz, Robert S.; Daida, Hiroyuki

    2016-01-01

    Background Several animal models have facilitated the evaluation and pathological understanding of atherosclerosis, but a definitive animal model of coronary atherosclerosis is not available. We therefore developed low density lipoprotein receptor knockout (LDLR-KO) pigs with hypercholesterolemia, a model which rapidly developed coronary atherosclerosis following balloon injury. Methods and Results We deleted LDLR exon regions from cultured porcine fetal fibroblasts and cloned LDLR knockout (LDLR-KO) embryos microinjecting fetal fibroblast nuclei into enucleated oocytes. Twelve LDLR-KO pigs were fed a 2.0% cholesterol and 20% fat diet. Baseline serum LDL cholesterol level was 510.0±86.1 mg/dL. Balloon injury was created in 46 coronary segments and necropsy were obtained 2, 4, 8 and 12 weeks later. Coronary artery sections were reviewed to evaluate lesion progression. We found lipid accumulation with foam cells and inflammatory cells beginning four weeks after balloon injury. The mean ratio of macrophages to plaque area was significantly higher in the four- weeks and eight-week animals compared with those at 2-weeks (8.79% ± 5.98% and 17.00% ± 10.38% vs. 1.14% ± 1.88%, P < 0.0001). At 12 weeks the ratio decreased toward the level at 2 week level (4.00% ± 4.56%, P = 0.66 vs. baseline). Advanced coronary atherosclerotic lesions contained lipid pools at eight-weeks with fibrous components beginning at 12 weeks. Conclusions We developed a model of rapid coronary atherosclerosis using LDLR KO pigs with balloon injury. This model may be useful for preclinical evaluation of medication or devices, and may also help investigate mechanisms of plaque progression. PMID:27631974

  7. 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.

  8. Inflammatory bone loss in experimental periodontitis induced by Aggregatibacter actinomycetemcomitans in interleukin-1 receptor antagonist knockout mice.

    PubMed

    Izawa, A; Ishihara, Y; Mizutani, H; Kobayashi, S; Goto, H; Okabe, E; Takeda, H; Ozawa, Y; Kamiya, Y; Sugita, Y; Kubo, K; Kamei, H; Kikuchi, T; Mitani, A; Hayashi, J; Nishihara, T; Maeda, H; Noguchi, T

    2014-05-01

    The interleukin-1 receptor antagonist (IL-1Ra) binds to IL-1 receptors and inhibits IL-1 activity. However, it is not clear whether IL-1Ra plays a protective role in periodontal disease. This study was undertaken to compare experimental periodontitis induced by Aggregatibacter actinomycetemcomitans in IL-1Ra knockout (KO) mice and wild-type (WT) mice. Computed tomography (CT) analysis and hematoxylin-and-eosin (H&E) and tartrate-resistant acid phosphatase (TRAP) staining were performed. In addition, osteoblasts were isolated; the mRNA expression of relevant genes was assessed by real-time quantitative PCR (qPCR); and calcification was detected by Alizarin Red staining. Infected IL-1Ra KO mice exhibited elevated (P, <0.05) levels of antibody against A. actinomycetemcomitans, bone loss in furcation areas, and alveolar fenestrations. Moreover, protein for tumor necrosis factor alpha (TNF-α) and IL-6, mRNA for macrophage colony-stimulating factor (M-CSF), and receptor activator of NF-κB ligand (RANKL) in IL-1Ra KO mouse osteoblasts stimulated with A. actinomycetemcomitans were increased (P, <0.05) compared to in WT mice. Alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OCN)/bone gla protein (BGP), and runt-related gene 2 (Runx2) mRNA levels were decreased (P, <0.05). IL-1α mRNA expression was increased, and calcification was not observed, in IL-1 Ra KO mouse osteoblasts. In brief, IL-1Ra deficiency promoted the expression of inflammatory cytokines beyond IL-1 and altered the expression of genes involved in bone resorption in A. actinomycetemcomitans-infected osteoblasts. Alterations consistent with rapid bone loss in infected IL-Ra KO mice were also observed for genes expressed in bone formation and calcification. In short, these data suggest that IL-1Ra may serve as a potential therapeutic drug for periodontal disease.

  9. Inflammatory bone loss in experimental periodontitis induced by Aggregatibacter actinomycetemcomitans in interleukin-1 receptor antagonist knockout mice.

    PubMed

    Izawa, A; Ishihara, Y; Mizutani, H; Kobayashi, S; Goto, H; Okabe, E; Takeda, H; Ozawa, Y; Kamiya, Y; Sugita, Y; Kubo, K; Kamei, H; Kikuchi, T; Mitani, A; Hayashi, J; Nishihara, T; Maeda, H; Noguchi, T

    2014-05-01

    The interleukin-1 receptor antagonist (IL-1Ra) binds to IL-1 receptors and inhibits IL-1 activity. However, it is not clear whether IL-1Ra plays a protective role in periodontal disease. This study was undertaken to compare experimental periodontitis induced by Aggregatibacter actinomycetemcomitans in IL-1Ra knockout (KO) mice and wild-type (WT) mice. Computed tomography (CT) analysis and hematoxylin-and-eosin (H&E) and tartrate-resistant acid phosphatase (TRAP) staining were performed. In addition, osteoblasts were isolated; the mRNA expression of relevant genes was assessed by real-time quantitative PCR (qPCR); and calcification was detected by Alizarin Red staining. Infected IL-1Ra KO mice exhibited elevated (P, <0.05) levels of antibody against A. actinomycetemcomitans, bone loss in furcation areas, and alveolar fenestrations. Moreover, protein for tumor necrosis factor alpha (TNF-α) and IL-6, mRNA for macrophage colony-stimulating factor (M-CSF), and receptor activator of NF-κB ligand (RANKL) in IL-1Ra KO mouse osteoblasts stimulated with A. actinomycetemcomitans were increased (P, <0.05) compared to in WT mice. Alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OCN)/bone gla protein (BGP), and runt-related gene 2 (Runx2) mRNA levels were decreased (P, <0.05). IL-1α mRNA expression was increased, and calcification was not observed, in IL-1 Ra KO mouse osteoblasts. In brief, IL-1Ra deficiency promoted the expression of inflammatory cytokines beyond IL-1 and altered the expression of genes involved in bone resorption in A. actinomycetemcomitans-infected osteoblasts. Alterations consistent with rapid bone loss in infected IL-Ra KO mice were also observed for genes expressed in bone formation and calcification. In short, these data suggest that IL-1Ra may serve as a potential therapeutic drug for periodontal disease. PMID:24566623

  10. Estrogen receptor function as revealed by knockout studies: neuroendocrine and behavioral aspects.

    PubMed

    Rissman, E F; Wersinger, S R; Taylor, J A; Lubahn, D B

    1997-06-01

    Estrogens are an important class of steroid hormones, involved in the development of brain, skeletal, and soft tissues. These hormones influence adult behaviors, endocrine state, and a host of other physiological functions. Given the recent cloning of a second estrogen receptor (ER) cDNA (the ER beta), work on alternate spliced forms of ER alpha, and the potential for membrane estrogen receptors, an animal with a null background for ER alpha function is invaluable for distinguishing biological responses of estrogens working via the ER alpha protein and those working via another ER protein. Data generated to date, and reviewed here, indicate that there are profound ramifications of the ER alpha disruption on behavior and neuroendocrine function. First, data on plasma levels of estradiol (E2), testosterone (T), and luteinizing hormone (LH) in wild-type (WT) versus ER alpha- mice confirm that ER alpha is essential in females for normal regulation of the hypothalamic-pituitary gonadal axis. Second, ovariectomized female ER alpha- mice do not display sexual receptivity when treated with a hormonal regime of estrogen and progesterone that induces receptivity in WT littermates. Finally, male sexual behaviors are disrupted in ER alpha- animals. Given decades of data on these topics our findings may seem self-evident. However, these data represent the most direct test currently possible of the specific role of the ER alpha protein on behavior and neuroendocrinology. The ER alpha- mouse can be used to ascertain the specific functions of ER alpha, to suggest functions for the other estrogen receptors, and to study indirect effects of ER alpha on behavior via actions on other receptors, neurotransmitters, and neuropeptides.

  11. Knockout of Angiotensin AT2 receptors accelerates healing but impairs quality

    PubMed Central

    Faghih, Mahya; Hosseini, Sayed M.; Smith, Barbara; Ansari, Amir Mehdi.; Lay, Frank; Ahmed, Ali Karim; Inagami, Tedashi; Marti, Guy P.; Harmon, John W.; Walston, Jeremy D.; Abadir, Peter M.

    2015-01-01

    Wounds are among the most common, painful, debilitating and costly conditions in older adults. Disruption of the angiotensin type 1 receptors (AT1R), has been associated with impaired wound healing, suggesting a critical role for AT1R in this repair process. Biological functions of angiotensin type 2 receptors (AT2R) are less studied. We investigated effects of genetically disrupting AT2R on rate and quality of wound healing. Our results suggest that AT2R effects on rate of wound closure depends on the phase of wound healing. We observed delayed healing during early phase of wound healing (inflammation). An accelerated healing rate was seen during later stages (proliferation and remodeling). By day 12, fifty percent of AT2R−/− mice had complete wound closure as compared to none in either C57/BL6 or AT1R−/− mice. There was a significant increase in AT1R, TGFβ1 and TGFβ2 expression during the proliferative and remodeling phases in AT2R−/− mice. Despite the accelerated closure rate, AT2R−/− mice had more fragile healed skin. Our results suggest that in the absence of AT2R, wound healing rate is accelerated, but yielded worse skin quality. Elucidating the contribution of both of the angiotensin receptors may help fine tune future intervention aimed at wound repair in older individuals. PMID:26727887

  12. 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

  13. Upregulation of Cannabinoid Type 1 Receptors in Dopamine D2 Receptor Knockout Mice Is Reversed by Chronic Forced Ethanol Consumption

    SciTech Connect

    Thanos, P.K.; Wang, G.; Thanos, P.K.; Gopez, V.; Delis, F.; Michaelides, M.; Grand, D.K.; Wang, G.-J.; Kunos, G.; Volkow, N.D.

    2011-01-01

    The anatomical proximity of the cannabinoid type 1 (CNR1/CB1R) and the dopamine D2 receptors (DRD2), their ability to form CB1R-DRD2 heteromers, their opposing roles in locomotion, and their involvement in ethanol's reinforcing and addictive properties prompted us to study the levels and distribution of CB1R after chronic ethanol intake, in the presence and absence of DRD2. We monitored the drinking patterns and locomotor activity of Drd2+/+ and Drd2-/- mice consuming either water or a 20% (v/v) ethanol solution (forced ethanol intake) for 6 months and used the selective CB1 receptor antagonist [{sup 3}H]SR141716A to quantify CB1R levels in different brain regions with in vitro receptor autoradiography. We found that the lack of DRD2 leads to a marked upregulation (approximately 2-fold increase) of CB1R in the cerebral cortex, the caudate-putamen, and the nucleus accumbens, which was reversed by chronic ethanol intake. The results suggest that DRD2-mediated dopaminergic neurotransmission and chronic ethanol intake exert an inhibitory effect on cannabinoid receptor expression in cortical and striatal regions implicated in the reinforcing and addictive properties of ethanol.

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. Hippocampal neurofibromin and amyloid precursor protein expression in dopamine D3 receptor knock-out mice following passive avoidance conditioning.

    PubMed

    D'Amico, Agata Grazia; Castorina, Alessandro; Leggio, Gian Marco; Drago, Filippo; D'Agata, Velia

    2013-03-01

    Passive avoidance (PA) conditioning is a fear motivated task able to initiate a cascade of altered gene expression within the hippocampus, a structure critical to learning and memory. We have previously shown that neurofibromin (NF1) and amyloid precursor protein (APP), two genes implicated in cognitive function, are differentially expressed in brain of dopamine D3 receptor knock-out mice (D(3)R(-/-)), suggesting that the receptor might have a role in their trascriptional regulation. Here in this study, we hypothesized that during acquisition of PA conditioning the expression of NF1 and APP genes could be influenced by D(3)Rs. To address this issue, we analyzed the expression of NF1 and APP in the hippocampus of both wild-type (WT) and D(3)R(-/-) mice subjected to the single trial step-through PA paradigm. Our finding demonstrated that (1) D(3)R(-/-) mice exhibit increased cognitive performance as compared to WT mice in the step-through PA trial; (2) acquisition of PA increased D(3)R and NF1, but not APP expression in WT mice hippocampus; (3) PA-driven NF1 induction in WT was abrogated in D(3)R(-/-) mice and finally that (4) the heightened basal APP expression observed in naive D(3)R(-/-) mice was totally reversed by acquisition of PA. In conclusion, the present finding show for the first time that both D(3)R and NF1 genes are upregulated following PA conditioning and suggest that hippocampal D(3)Rs might be relevant to NF1 transcriptional regulation in the hippocampus.

  19. Colonic mucosal DNA methylation, immune response, and microbiome patterns in Toll-like receptor 2-knockout mice

    PubMed Central

    Kellermayer, Richard; Dowd, Scot E.; Harris, R. Alan; Balasa, Alfred; Schaible, Tiffany D.; Wolcott, Randy D.; Tatevian, Nina; Szigeti, Reka; Li, Zhijie; Versalovic, James; Smith, C. Wayne

    2011-01-01

    The connection between intestinal microbiota and host physiology is increasingly becoming recognized. The details of this dynamic interaction, however, remain to be explored. Toll-like receptor 2 (Tlr2) is important for its role in bacterial recognition, intestinal inflammation, and obesity-related metabolic changes. Therefore, we sought to determine the epigenomic and metagenomic consequences of Tlr2 deficiency in the colonic mucosa of mice to gain insights into biological pathways that shape the interface between the gut microbiota and the mammalian host. Colonic mucosa from wild type (WT) and Tlr2−/− C57BL/6 mice was interrogated by microarrays specific for DNA methylation and gene expression. The mucosal microbiome was studied by next-generation pyrosequencing of bacterial 16S rRNA. The expression of genes involved in immune processes was significantly modified by the absence of Tlr2, a number of which correlated with DNA methylation changes. The epigenomic and transcriptomic modifications associated with alteration in mucosal microbial composition. Several bacterial species, including members of the Firmicutes were significantly different in abundance between WT and Tlr2−/− animals. This manuscript highlights the intimate interrelationships between expression of immune-related genes and immunity pathways in the host with compositional and functional differences of the mammalian microbiome.—Kellermayer, R., Dowd, S. E., Harris, R. A., Balasa, A., Schaible, T. D., Wolcott, R. D., Tatevian, N., Szigeti, R., Li, Z., Versalovic, J., Smith, C. W. Colonic mucosal DNA methylation, immune response, and microbiome patterns in Toll-like receptor 2-knockout mice. PMID:21228220

  20. Progesterone receptor knockout mice have an improved glucose homeostasis secondary to -cell proliferation

    NASA Astrophysics Data System (ADS)

    Picard, Frédéric; Wanatabe, Mitsuhiro; Schoonjans, Kristina; Lydon, John; O'Malley, Bert W.; Auwerx, Johan

    2002-11-01

    Gestational diabetes coincides with elevated circulating progesterone levels. We show that progesterone accelerates the progression of diabetes in female db/db mice. In contrast, RU486, an antagonist of the progesterone receptor (PR), reduces blood glucose levels in both female WT and db/db mice. Furthermore, female, but not male, PR-/- mice had lower fasting glycemia than PR+/+ mice and showed higher insulin levels on glucose injection. Pancreatic islets from female PR-/- mice were larger and secreted more insulin consequent to an increase in -cell mass due to an increase in -cell proliferation. These findings demonstrate an important role of progesterone signaling in insulin release and pancreatic function and suggest that it affects the susceptibility to diabetes.

  1. Small heterodimer partner overexpression partially protects against liver tumor development in farnesoid X receptor knockout mice

    SciTech Connect

    Li, Guodong; Kong, Bo; Zhu, Yan; Zhan, Le; Williams, Jessica A.; Tawfik, Ossama; Kassel, Karen M.; Luyendyk, James P.; Wang, Li; Guo, Grace L.

    2013-10-15

    Farnesoid X receptor (FXR, Nr1h4) and small heterodimer partner (SHP, Nr0b2) are nuclear receptors that are critical to liver homeostasis. Induction of SHP serves as a major mechanism of FXR in suppressing gene expression. Both FXR{sup −/−} and SHP{sup −/−} mice develop spontaneous hepatocellular carcinoma (HCC). SHP is one of the most strongly induced genes by FXR in the liver and is a tumor suppressor, therefore, we hypothesized that deficiency of SHP contributes to HCC development in the livers of FXR{sup −/−} mice and therefore, increased SHP expression in FXR{sup −/−} mice reduces liver tumorigenesis. To test this hypothesis, we generated FXR{sup −/−} mice with overexpression of SHP in hepatocytes (FXR{sup −/−}/SHP{sup Tg}) and determined the contribution of SHP in HCC development in FXR{sup −/−} mice. Hepatocyte-specific SHP overexpression did not affect liver tumor incidence or size in FXR{sup −/−} mice. However, SHP overexpression led to a lower grade of dysplasia, reduced indicator cell proliferation and increased apoptosis. All tumor-bearing mice had increased serum bile acid levels and IL-6 levels, which was associated with activation of hepatic STAT3. In conclusion, SHP partially protects FXR{sup −/−} mice from HCC formation by reducing tumor malignancy. However, disrupted bile acid homeostasis by FXR deficiency leads to inflammation and injury, which ultimately results in uncontrolled cell proliferation and tumorigenesis in the liver. - Highlights: • SHP does not prevent HCC incidence nor size in FXR KO mice but reduces malignancy. • Increased SHP promotes apoptosis. • Bile acids and inflammation maybe critical for HCC formation with FXR deficiency.

  2. Vasopressin 1b receptor knock-out impairs memory for temporal order.

    PubMed

    DeVito, Loren M; Konigsberg, Rachael; Lykken, Christine; Sauvage, Magdalena; Young, W Scott; Eichenbaum, Howard

    2009-03-01

    Mice lacking a functional vasopressin 1b receptor (Avpr1b) display decreased levels of aggression and social memory. Here, we used Avpr1b-knock-out (Avpr1b(-/-)) mice to examine whether an abnormality of this receptor results in specific cognitive deficits in the domain of hippocampal function. Avpr1b(-/-) mice were deficient in sociability and in detecting social novelty, extending previous findings of impairment in social recognition in these mutants. Avpr1b(-/-) mice could recognize previously explored objects and remember where they were experienced, but they were impaired in remembering the temporal order of presentation of those objects. Consistent with this finding, Avpr1b(-/-) mice were also impaired on an object-odor paired associate task that involved a temporal discontiguity between the associated elements. Finally, Avpr1b(-/-) mice performed normally in learning a set of overlapping odor discriminations and could infer relationships among odors that were only indirectly associated (i.e., transitive inference), indicating intact relational memory. The Avpr1b is expressed at much higher levels than any other part of the brain in the pyramidal cells of hippocampal CA2 area, a subfield of the hippocampus that has physiological and genetic properties that distinguish it from subfields CA1 and CA3. The combined results suggest that the Avpr1b, perhaps in CA2, may play a highly specific role in social behavior and episodic memory. Because schizophrenia and bipolar disorder are associated with a unique pathology in CA2 and impairments in both social behavior and episodic memory, this animal model could provide insights into the etiology of these disorders.

  3. Knockout of the aryl hydrocarbon receptor results in distinct hepatic and renal phenotypes in rats and mice

    SciTech Connect

    Harrill, Joshua A.; Hukkanen, Renee R.; Lawson, Marie; Martin, Greg; Gilger, Brian; Soldatow, Valerie; LeCluyse, Edward L.; Budinsky, Robert A.; Rowlands, J. Craig; Thomas, Russell S.

    2013-10-15

    The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor which plays a role in the development of multiple tissues and is activated by a large number of ligands, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In order to examine the roles of the AHR in both normal biological development and response to environmental chemicals, an AHR knockout (AHR-KO) rat model was created and compared with an existing AHR-KO mouse. AHR-KO rats harboring either 2-bp or 29-bp deletion mutation in exon 2 of the AHR were created on the Sprague–Dawley genetic background using zinc-finger nuclease (ZFN) technology. Rats harboring either mutation type lacked expression of AHR protein in the liver. AHR-KO rats were also insensitive to thymic involution, increased hepatic weight and the induction of AHR-responsive genes (Cyp1a1, Cyp1a2, Cyp1b1, Ahrr) following acute exposure to 25 μg/kg TCDD. AHR-KO rats had lower basal expression of transcripts for these genes and also accumulated ∼ 30–45-fold less TCDD in the liver at 7 days post-exposure. In untreated animals, AHR-KO mice, but not AHR-KO rats, had alterations in serum analytes indicative of compromised hepatic function, patent ductus venosus of the liver and persistent hyaloid arteries in the eye. AHR-KO rats, but not AHR-KO mice, displayed pathological alterations to the urinary tract: bilateral renal dilation (hydronephrosis), secondary medullary tubular and uroepithelial degenerative changes and bilateral ureter dilation (hydroureter). The present data indicate that the AHR may play significantly different roles in tissue development and homeostasis and toxicity across rodent species. - Highlights: • An AHR knockout rat was generated on a Sprague–Dawley outbred background. • AHR-KO rats lack expression of AHR protein. • AHR-KO rats are insensitive to TCDD-mediated effects. • Data suggests difference in the role of AHR in tissue development of rats and mice. • Abnormalities in vascular

  4. Ovariectomy-mediated impairment of spatial working memory, but not reference memory, is attenuated by the knockout of the dopamine D(3) receptor in female mice.

    PubMed

    Cao, Fang; Zhang, Hongbo; Meng, Xia; Feng, Jiali; Li, Teng; Wei, Shuguang; Li, Shengbin

    2013-06-15

    Memory modulation is implemented through many different factors, such as the dopaminergic system and ovarian hormones. Alterations of these factors may cause memory behavior changes. In the current study, dopamine D3 receptor mutant (D3(-/-)) female mice and their wild-type (WT) controls were used and randomly assigned to ovariectomized (OVX) and sham-operated (Sham) groups (four groups: WT-OVX, WT-Sham, D3(-/-)-OVX, and D3(-/-)-Sham). The possibility of co-effects of the dopamine D3 receptor and ovarian hormones on cognitive behavior was then examined. The spatial reference memory (SRM) and the motivation to escape from the water maze were not affected by either knockout of the dopamine D3 receptor or ovariectomy. Knockout of the dopamine D3 receptor had no effect on spatial working memory (SWM) in the Sham groups, but had a positive effect on SWM in the OVX groups. While an ovariectomy inhibited SWM in WT mice, it had no effect on SWM in D3(-/-) mice. These data suggested that ovarian hormone deprivation may induce spatial working memory decline, and blockade of D3 receptors could ameliorate this impairment. Based on our findings, there may be specific molecular changes responsible for the function of D3 receptors in modulating information processes under conditions of ovarian hormone deficit, which need to be further elucidated.

  5. Development of schemas revealed by prior experience and NMDA receptor knock-out

    PubMed Central

    Dragoi, George; Tonegawa, Susumu

    2013-01-01

    Prior experience accelerates acquisition of novel, related information through processes like assimilation into mental schemas, but the underlying neuronal mechanisms are poorly understood. We investigated the roles that prior experience and hippocampal CA3 N-Methyl-D-aspartate receptor (NMDAR)-dependent synaptic plasticity play in CA1 place cell sequence encoding and learning during novel spatial experiences. We found that specific representations of de novo experiences on linear environments were formed on a framework of pre configured network activity expressed in the preceding sleep and were rapidly, flexibly adjusted via NMDAR-dependent activity. This prior experience accelerated encoding of subsequent experiences on contiguous or isolated novel tracks, significantly decreasing their NMDAR-dependence. Similarly, de novo learning of an alternation task was facilitated by CA3 NMDARs; this experience accelerated subsequent learning of related tasks, independent of CA3 NMDARs, consistent with a schema-based learning. These results reveal the existence of distinct neuronal encoding schemes which could explain why hippocampal dysfunction results in anterograde amnesia while sparing recollection of old, schema-based memories. DOI: http://dx.doi.org/10.7554/eLife.01326.001 PMID:24327561

  6. Altered enteroendocrine cell expression in T cell receptor alpha chain knock-out mice.

    PubMed

    Rubin, D C; Zhang, H; Qian, P; Lorenz, R G; Hutton, K; Peters, M G

    2000-10-15

    Mice lacking T cell receptor alpha chain (TCRalpha(-/-)) develop inflammation of the colon. We have examined the effect of this inflammation on the colonic epithelium by studying markers of epithelial cuff, enteroendocrine, and immune cell differentiation. Using immunohistochemical techniques, colons were compared in normal C57/BL6 and murine TCR alpha(-/-) mice aged 2 and 3 weeks and 3-11 months. TCR alpha(-/-) mice aged 3-11 months had histologic evidence of inflammation with increased expression of CD45, CD4+, CD8+, and B220+ cells and a decrease in expression of IgA+ cells. There was a decrease in the number of cholecystokinin, serotonin, and neurotensin enteroendocrine expressing cells in the colon of TCR alpha(-/-) mice. These changes were not present in 2-3-week-old suckling/weaning mice. In contrast, peptide tyrosine tyrosine (PYY), glucagon-like peptide-1, and gastrin expression did not change and small intestinal enteroendocrine cells remained unaltered. The change in colonic enteroendocrine cell expression appears to be a specific response, since only a subset of these cells was altered, and the epithelium was intact by histologic analysis. The absence of functional T cells in TCR alpha(-/-) colon has a marked effect on differentiation of a specific subpopulation of enteroendocrine cells, prior to loss of integrity of the epithelium. PMID:11054861

  7. Endothelium dysfunction in LDL receptor knockout mice: a role for H2O2

    PubMed Central

    Rabelo, Luíza A; Cortes, Steyner F; Alvarez-Leite, Jacqueline I; Lemos, Virgínia S

    2003-01-01

    In this study, the role of endogenous H2O2 as an endothelium-dependent relaxant factor was characterised in aortas from C57BL/6J and LDL receptor-deficient mice (LDLR−/−). Aortic rings from LDLR−/− mice showed impaired endothelium-dependent relaxation to acetylcholine (ACh; 0.001–100 μM) and to the Ca2+ ionophore A23187 (0.001–3 μM) compared with aortic rings from control mice. Endothelium-independent relaxation produced by the NO donor, 3-morpholino-sydnonimine (SIN-1) was not different between strains. Pretreatment of vessels with L-NNA (100 μM) or L-NNA (100 μM) plus L-NAME (300 μM) plus haemoglobin (10 μM) markedly decreased, but did not abolish the relaxation to ACh in control mice. In the aortas from LDLR−/− mice treated with L-NNA (100 μM), ACh induced a contractile effect. Catalase (800 and 2400 U ml−1) shifted to the right the endothelium-dependent relaxation to ACh in aortas from control but not from LDLR−/− mice. Aminotriazole (50 mM), which inhibits catalase, abolished its effect on control mice. Treatment of vessels with L-NNA and catalase abolished vasorelaxation induced by ACh. Indomethacin (10 μM) did not modify the concentration–response curve to ACh. Superoxide dismutase (300 U ml−1) did not change ACh-induced relaxation in both strains. Exogenous H2O2 produced a concentration-dependent relaxation in endothelium-denuded aortic rings, which was not different between strains. It is concluded that H2O2 greatly contributes to relaxation to ACh in aorta from control mice. Endothelial-dependent relaxation to ACh is impaired in LDLR−/− mice. Reduced biosynthesis or increased inactivation of H2O2 is the possible mechanism responsible for endothelial dysfunction in aortas of atherosclerosis-susceptible LDLR−/− mice. PMID:12711621

  8. Aryl hydrocarbon receptor knock-out exacerbates choroidal neovascularization via multiple pathogenic pathways.

    PubMed

    Choudhary, Mayur; Kazmin, Dmitri; Hu, Peng; Thomas, Russell S; McDonnell, Donald P; Malek, Goldis

    2015-01-01

    The aryl hydrocarbon receptor (AhR) is a heterodimeric transcriptional regulator with pleiotropic functions in xenobiotic metabolism and detoxification, vascular development and cancer. Herein, we report a previously undescribed role for the AhR signalling pathway in the pathogenesis of the wet, neovascular subtype of age-related macular degeneration (AMD), the leading cause of vision loss in the elderly in the Western world. Comparative analysis of gene expression profiles of aged AhR(-/-) and wild-type (wt) mice, using high-throughput RNA sequencing, revealed differential modulation of genes belonging to several AMD-related pathogenic pathways, including inflammation, angiogenesis and extracellular matrix regulation. To investigate AhR regulation of these pathways in wet AMD, we experimentally induced choroidal neovascular lesions in AhR(-/-) mice and found that they measured significantly larger in area and volume compared to age-matched wt mice. Furthermore, these lesions displayed a higher number of ionized calcium-binding adaptor molecule 1-positive (Iba1(+) ) microglial cells and a greater amount of collagen type IV deposition, events also seen in human wet AMD pathology specimens. Consistent with our in vivo observations, AhR knock-down was sufficient to increase choroidal endothelial cell migration and tube formation in vitro. Moreover, AhR knock-down caused an increase in collagen type IV production and secretion in both retinal pigment epithelial (RPE) and choroidal endothelial cell cultures, increased expression of angiogenic and inflammatory molecules, including vascular endothelial growth factor A (VEGFA) and chemokine (C-C motif) ligand 2 (CCL2) in RPE cells, and increased expression of secreted phosphoprotein 1 (SPP1) and transforming growth factor-β1 (TGFβ1) in choroidal endothelial cells. Collectively, our findings identify AhR as a regulator of multiple pathogenic pathways in experimentally induced choroidal neovascularization, findings that

  9. Aryl hydrocarbon receptor knock-out exacerbates choroidal neovascularization via multiple pathogenic pathways

    PubMed Central

    Choudhary, Mayur; Kazmin, Dmitri; Hu, Peng; Thomas, Russell S; McDonnell, Donald P; Malek, Goldis

    2015-01-01

    The aryl hydrocarbon receptor (AhR) is a heterodimeric transcriptional regulator with pleiotropic functions in xenobiotic metabolism and detoxification, vascular development and cancer. Herein, we report a previously undescribed role for the AhR signalling pathway in the pathogenesis of the wet, neovascular subtype of age-related macular degeneration (AMD), the leading cause of vision loss in the elderly in the Western world. Comparative analysis of gene expression profiles of aged AhR−/− and wild-type (wt) mice, using high-throughput RNA sequencing, revealed differential modulation of genes belonging to several AMD-related pathogenic pathways, including inflammation, angiogenesis and extracellular matrix regulation. To investigate AhR regulation of these pathways in wet AMD, we experimentally induced choroidal neovascular lesions in AhR−/− mice and found that they measured significantly larger in area and volume compared to age-matched wt mice. Furthermore, these lesions displayed a higher number of ionized calcium-binding adaptor molecule 1-positive (Iba1+) microglial cells and a greater amount of collagen type IV deposition, events also seen in human wet AMD pathology specimens. Consistent with our in vivo observations, AhR knock-down was sufficient to increase choroidal endothelial cell migration and tube formation in vitro. Moreover, AhR knock-down caused an increase in collagen type IV production and secretion in both retinal pigment epithelial (RPE) and choroidal endothelial cell cultures, increased expression of angiogenic and inflammatory molecules, including vascular endothelial growth factor A (VEGFA) and chemokine (C–C motif) ligand 2 (CCL2) in RPE cells, and increased expression of secreted phosphoprotein 1 (SPP1) and transforming growth factor-β1 (TGFβ1) in choroidal endothelial cells. Collectively, our findings identify AhR as a regulator of multiple pathogenic pathways in experimentally induced choroidal neovascularization, findings that

  10. Impact of growth hormone resistance on female reproductive function: new insights from growth hormone receptor knockout mice.

    PubMed

    Zaczek, Denise; Hammond, James; Suen, Lii; Wandji, Serge; Service, Darlene; Bartke, Andrzej; Chandrashekar, Varadaraj; Coschigano, Karen; Kopchick, John

    2002-10-01

    We examined multiple aspects of reproductive function in growth hormone receptor gene knockout (GHR-KO) and normal mice to clarify the role of growth hormone in female reproduction. In adult animals, estrous cycle duration was comparable in all mice housed individually but was significantly longer in group-housed GHR-KO females. Histological evaluation of ovaries of adult females at estrus showed that the numbers of preovulatory follicles and corpora lutea were significantly reduced in GHR-KO mice, as was the plasma estradiol level. The number of atretic preovulatory follicles was reduced in GHR gene-ablated animals. Although reverse transcription polymerase chain reaction analysis revealed reduced ovarian insulin-like growth factor I (IGF-I) mRNA expression in GHR-KO females, the expression of several steroidogenic enzyme mRNAs did not differ between groups. The numbers of active corpora lutea and uterine implantation sites were reduced in GHR-KO females at Day 7 of gestation. When young females were mated to normal males, latency to first mating and age of the female at first mating were significantly delayed in GHR-KO females, but maternal age at first conception was similar between groups. Significantly fewer virgin GHR-KO females exhibited pseudopregnancies when initially placed with vasectomized normal males than did normal female counterparts. Growth hormone resistance and IGF-I insufficiency negatively impacted 1) follicular development/ovulation rate, 2) sexual maturation, 3) production of and responsiveness to pheromonal signals, and 4) the ability of virgin females to respond to coitus by activation of luteal function. Although GHR-KO female mice are fertile, they exhibit quantitative deficits in various parameters of reproductive function.

  11. Knockout of NMDA-receptors from parvalbumin interneurons sensitizes to schizophrenia-related deficits induced by MK-801

    PubMed Central

    Bygrave, A M; Masiulis, S; Nicholson, E; Berkemann, M; Barkus, C; Sprengel, R; Harrison, P J; Kullmann, D M; Bannerman, D M; Kätzel, D

    2016-01-01

    It has been suggested that a functional deficit in NMDA-receptors (NMDARs) on parvalbumin (PV)-positive interneurons (PV-NMDARs) is central to the pathophysiology of schizophrenia. Supportive evidence come from examination of genetically modified mice where the obligatory NMDAR-subunit GluN1 (also known as NR1) has been deleted from PV interneurons by Cre-mediated knockout of the corresponding gene Grin1 (Grin1ΔPV mice). Notably, such PV-specific GluN1 ablation has been reported to blunt the induction of hyperlocomotion (a surrogate for psychosis) by pharmacological NMDAR blockade with the non-competitive antagonist MK-801. This suggests PV-NMDARs as the site of the psychosis-inducing action of MK-801. In contrast to this hypothesis, we show here that Grin1ΔPV mice are not protected against the effects of MK-801, but are in fact sensitized to many of them. Compared with control animals, Grin1ΔPVmice injected with MK-801 show increased stereotypy and pronounced catalepsy, which confound the locomotor readout. Furthermore, in Grin1ΔPVmice, MK-801 induced medial-prefrontal delta (4 Hz) oscillations, and impaired performance on tests of motor coordination, working memory and sucrose preference, even at lower doses than in wild-type controls. We also found that untreated Grin1ΔPVmice are largely normal across a wide range of cognitive functions, including attention, cognitive flexibility and various forms of short-term memory. Taken together these results argue against PV-specific NMDAR hypofunction as a key starting point of schizophrenia pathophysiology, but support a model where NMDAR hypofunction in multiple cell types contribute to the disease. PMID:27070406

  12. Knockout of the aryl hydrocarbon receptor results in distinct hepatic and renal phenotypes in rats and mice.

    PubMed

    Harrill, Joshua A; Hukkanen, Renee R; Lawson, Marie; Martin, Greg; Gilger, Brian; Soldatow, Valerie; Lecluyse, Edward L; Budinsky, Robert A; Rowlands, J Craig; Thomas, Russell S

    2013-10-15

    The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor which plays a role in the development of multiple tissues and is activated by a large number of ligands, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In order to examine the roles of the AHR in both normal biological development and response to environmental chemicals, an AHR knockout (AHR-KO) rat model was created and compared with an existing AHR-KO mouse. AHR-KO rats harboring either 2-bp or 29-bp deletion mutation in exon 2 of the AHR were created on the Sprague-Dawley genetic background using zinc-finger nuclease (ZFN) technology. Rats harboring either mutation type lacked expression of AHR protein in the liver. AHR-KO rats were also insensitive to thymic involution, increased hepatic weight and the induction of AHR-responsive genes (Cyp1a1, Cyp1a2, Cyp1b1, Ahrr) following acute exposure to 25 μg/kg TCDD. AHR-KO rats had lower basal expression of transcripts for these genes and also accumulated ~30-45-fold less TCDD in the liver at 7 days post-exposure. In untreated animals, AHR-KO mice, but not AHR-KO rats, had alterations in serum analytes indicative of compromised hepatic function, patent ductus venosus of the liver and persistent hyaloid arteries in the eye. AHR-KO rats, but not AHR-KO mice, displayed pathological alterations to the urinary tract: bilateral renal dilation (hydronephrosis), secondary medullary tubular and uroepithelial degenerative changes and bilateral ureter dilation (hydroureter). The present data indicate that the AHR may play significantly different roles in tissue development and homeostasis and toxicity across rodent species. PMID:23859880

  13. Yellow wine polyphenolic compounds inhibit matrix metalloproteinase-2, -9 expression and improve atherosclerotic plaque in LDL-receptor-knockout mice.

    PubMed

    Zhai, Xiaoya; Chi, Jufang; Tang, Weiliang; Ji, Zheng; Zhao, Fei; Jiang, Chengjian; Lv, Haitao; Guo, Hangyuan

    2014-01-01

    Many epidemiological studies have strongly suggested an inverse correlation between dietary polyphenol consumption and reduced risks of cardiovascular diseases. Yellow rice wine is a Chinese specialty and one of the three most ancient wines in the world (Shaoxing rice wine, beer, and grape wine). There is a large amount of polyphenol substances in yellow rice wine. This experiment was designed to study the potential beneficial effects of yellow wine polyphenolic compounds (YWPC) from yellow rice wine on progression of atherosclerosis in vivo and to further explore its underlying mechanisms. Six-week-old male LDL-receptor-knockout mice were treated with high-fat diet to establish the mouse model with atherosclerosis. Animals received 10, 30, or 50 mg/kg per day of YWPC or 10 mg/kg per day rosuvastatin or water (vehicle) for 14 weeks. The results indicated that YWPC and rosuvastatin significantly decreased circulating total cholesterol and low-density lipoprotein cholesterol. Compared to the control group, the atherosclerosis lesion area in the rosuvastatin-intervention group and YWPC at doses of 10, 30, and 50 mg/kg per day intervention groups decreased by 74.14%, 18.51%, 40.09%, and 38.42%, respectively. YWPC and rosuvastatin decreased the expression and activity of matrix metalloproteinases (MMP)-2, 9, whereas the expression of the endogenous inhibitors of these proteins, namely, tissue inhibitors of matrix metalloproteinases (TIMP)-1, 2, increased when compared to the control group. It can be concluded that the YWPC is similar to the benefic effects of rosuvastatin on cardiovascular system. These effects may be attributed to their anti-atherosclerotic actions by lowering lipid and modulating the activity and expression of MMP-2, 9 and TIMP-1, 2.

  14. 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

  15. 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.

  16. Differential regulation of primary afferent input to spinal cord by muscarinic receptor subtypes delineated using knockout mice.

    PubMed

    Chen, Shao-Rui; Chen, Hong; Yuan, Wei-Xiu; Wess, Jürgen; Pan, Hui-Lin

    2014-05-16

    Stimulation of muscarinic acetylcholine receptors (mAChRs) inhibits nociceptive transmission at the spinal level. However, it is unclear how each mAChR subtype regulates excitatory synaptic input from primary afferents. Here we examined excitatory postsynaptic currents (EPSCs) of dorsal horn neurons evoked by dorsal root stimulation in spinal cord slices from wild-type and mAChR subtype knock-out (KO) mice. In wild-type mice, mAChR activation with oxotremorine-M decreased the amplitude of monosynaptic EPSCs in ∼67% of neurons but increased it in ∼10% of neurons. The inhibitory effect of oxotremorine-M was attenuated by the M2/M4 antagonist himbacine in the majority of neurons, and the remaining inhibition was abolished by group II/III metabotropic glutamate receptor (mGluR) antagonists in wild-type mice. In M2/M4 double-KO mice, oxotremorine-M inhibited monosynaptic EPSCs in significantly fewer neurons (∼26%) and increased EPSCs in significantly more neurons (33%) compared with wild-type mice. Blocking group II/III mGluRs eliminated the inhibitory effect of oxotremorine-M in M2/M4 double-KO mice. In M2 single-KO and M4 single-KO mice, himbacine still significantly reduced the inhibitory effect of oxotremorine-M. However, the inhibitory and potentiating effects of oxotremorine-M on EPSCs in M3 single-KO and M1/M3 double-KO mice were similar to those in wild-type mice. In M5 single-KO mice, oxotremorine-M failed to potentiate evoked EPSCs, and its inhibitory effect was abolished by himbacine. These findings indicate that activation of presynaptic M2 and M4 subtypes reduces glutamate release from primary afferents. Activation of the M5 subtype either directly increases primary afferent input or inhibits it through indirectly stimulating group II/III mGluRs. PMID:24695732

  17. Vaccination with recombinant adenoviruses expressing Ebola virus glycoprotein elicits protection in the interferon alpha/beta receptor knock-out mouse.

    PubMed

    O'Brien, Lyn M; Stokes, Margaret G; Lonsdale, Stephen G; Maslowski, David R; Smither, Sophie J; Lever, Mark S; Laws, Thomas R; Perkins, Stuart D

    2014-03-01

    The resistance of adult immunocompetent mice to infection with ebolaviruses has led to the development of alternative small animal models that utilise immunodeficient mice, for example the interferon α/β receptor knock-out mouse (IFNR(-/-)). IFNR(-/-) mice have been shown to be susceptible to infection with ebolaviruses by multiple routes but it is not known if this murine model is suitable for testing therapeutics that rely on the generation of an immune response for efficacy. We have tested recombinant adenovirus vectors for their ability to protect IFNR(-/-) mice from challenge with Ebola virus and have analysed the humoral response generated after immunisation. The recombinant vaccines elicited good levels of protection in the knock-out mouse and the antibody response in IFNR(-/-) mice was similar to that observed in vaccinated wild-type mice. These results indicate that the IFNR(-/-) mouse is a relevant small animal model for studying ebolavirus-specific therapeutics.

  18. Prevention of the polycystic ovarian phenotype and characterization of ovulatory capacity in the estrogen receptor-alpha knockout mouse.

    PubMed

    Couse, J F; Bunch, D O; Lindzey, J; Schomberg, D W; Korach, K S

    1999-12-01

    Ovarian-derived estradiol plays a critical endocrine role in the regulation of gonadotropin synthesis and secretion from the hypothalamic-pituitary axis. In turn, several para/autocrine effects of estrogen within the ovary are known, including increased ovarian weight, stimulation of granulosa cell growth, augmentation of FSH action, and attenuation of apoptosis. The estrogen receptor-alpha (ERalpha) is present in all three components of the hypothalamic-pituitary-ovarian axis of the mouse. In contrast, estrogen receptor-beta (ERbeta) is easily detectable in ovarian granulosa cells but is low to absent in the pituitary of the adult mouse. This distinct expression pattern for the two ERs suggests the presence of separate roles for each in the regulation of ovarian function. Herein, we definitively show that a lack of ERalpha in the hypothalamic-pituitary axis of the ERalpha-knockout (alphaERKO) mouse results in chronic elevation of serum LH and is the primary cause of the ovarian phenotype of polycystic follicles and anovulation. Prolonged treatment with a GnRH antagonist reduced serum LH levels and prevented the alphaERKO cystic ovarian phenotype. To investigate a direct role for ERalpha within the ovary, immature alphaERKO females were stimulated to ovulate with exogenous gonadotropins. Ovulatory capacity in the immature alphaERKO female was reduced compared with age-matched wild-type (14.5+/-2.9 vs. 40.6+/-2.6 oocytes/animal, respectively); however, oocytes collected from the alphaERKO were able to undergo successful in vitro fertilization. A similar discrepancy in oocyte yield was observed after superovulation of peripubertal (42 days) wild-type and alphaERKO females. In addition, ovaries from immature superovulated alphaERKO females possessed several ovulatory but unruptured follicles. Investigations of the possible reasons for the reduced number of ovulations in the alphaERKO included ribonuclease protection assays to assess the mRNA levels of several markers

  19. β-Arrestin-2 knockout prevents development of cellular μ-opioid receptor tolerance but does not affect opioid-withdrawal-related adaptations in single PAG neurons

    PubMed Central

    Connor, M; Bagley, E E; Chieng, B C; Christie, M J

    2015-01-01

    BACKGROUND AND PURPOSE Tolerance to the behavioural effects of morphine is blunted in β-arrestin-2 knockout mice, but opioid withdrawal is largely unaffected. The cellular mechanisms of tolerance have been studied in some neurons from β-arrestin-2 knockouts, but tolerance and withdrawal mechanisms have not been examined at the cellular level in periaqueductal grey (PAG) neurons, which are crucial for central tolerance and withdrawal phenomena. EXPERIMENTAL APPROACH μ-Opioid receptor (MOPr) inhibition of voltage-gated calcium channel currents (ICa) was examined by patch-clamp recordings from acutely dissociated PAG neurons from wild-type and β-arrestin-2 knockout mice treated chronically with morphine (CMT) or vehicle. Opioid withdrawal-induced activation of GABA transporter type 1 (GAT-1) currents was determined using perforated patch recordings from PAG neurons in brain slices. KEY RESULTS MOPr inhibition of ICa in PAG neurons was unaffected by β-arrestin-2 deletion. CMT impaired coupling of MOPrs to ICa in PAG neurons from wild-type mice, but this cellular tolerance was not observed in neurons from CMT β-arrestin-2 knockouts. However, β-arrestin-2 knockouts displayed similar opioid-withdrawal-induced activation of GAT-1 currents as wild-type PAG neurons. CONCLUSIONS AND IMPLICATIONS In β-arrestin-2 knockout mice, the central neurons involved in the anti-nociceptive actions of opioids also fail to develop cellular tolerance to opioids following chronic morphine. The results also provide the first cellular physiological evidence that opioid withdrawal is not disrupted by β-arrestin-2 deletion. However, the unaffected basal sensitivity to opioids in PAG neurons provides further evidence that changes in basal MOPr sensitivity cannot account for the enhanced acute nociceptive response to morphine reported in β-arrestin-2 knockouts. LINKED ARTICLES This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other

  20. Enhanced effects of amphetamine but reduced effects of the hallucinogen, 5-MeO-DMT, on locomotor activity in 5-HT(1A) receptor knockout mice: implications for schizophrenia.

    PubMed

    van den Buuse, Maarten; Ruimschotel, Emma; Martin, Sally; Risbrough, Victoria B; Halberstadt, Adam L

    2011-01-01

    Serotonin-1A (5-HT(1A)) receptors may play a role in schizophrenia and the effects of certain antipsychotic drugs. However, the mechanism of interaction of 5-HT(1A) receptors with brain systems involved in schizophrenia, remains unclear. Here we show that 5-HT(1A) receptor knockout mice display enhanced locomotor hyperactivity to acute treatment with amphetamine, a widely used animal model of hyperdopaminergic mechanisms in psychosis. In contrast, the effect of MK-801 on locomotor activity, modeling NMDA receptor hypoactivity, was unchanged in the knockouts. The effect of the hallucinogen 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) was markedly reduced in 5-HT(1A) receptor knockout mice. There were no changes in apomorphine-induced disruption of PPI, a model of sensory gating deficits seen in schizophrenia. Similarly, there were no major changes in density of dopamine transporters (DAT) or dopamine D(1) or D(2) receptors which could explain the behavioural changes observed in 5-HT(1A) receptor knockout mice. These results extend our insight into the possible role of these receptors in aspects of schizophrenia. As also suggested by previous studies using agonist and antagonist drugs, 5-HT(1A) receptors may play an important role in hallucinations and to modulate dopaminergic activity in the brain.

  1. Knockout of Toll-Like Receptors 2 and 4 Prevents Renal Ischemia-Reperfusion-Induced Cardiac Hypertrophy in Mice

    PubMed Central

    Trentin-Sonoda, Mayra; da Silva, Rogério Cirino; Kmit, Fernanda Vieira; Abrahão, Mariana Vieira; Monnerat Cahli, Gustavo; Brasil, Guilherme Visconde; Muzi-Filho, Humberto; Silva, Paulo André; Tovar-Moll, Fernanda Freire; Vieyra, Adalberto; Medei, Emiliano; Carneiro-Ramos, Marcela Sorelli

    2015-01-01

    We investigated whether the pathways linked to Toll-like receptors 2 and 4 (TLRs) are involved in renal ischemia-reperfusion (I/R)-induced cardiac hypertrophy. Wild type (WT) C57BL/6J, TLR2-/- and TLR4-/- mice were subjected to left kidney ischemia for 60 min followed by reperfusion for 5, 8, 12 and 15 days. Proton density magnetic resonance showed alterations in the injured kidney from WT mice, together with signs of parenchymal edema and higher levels of vimentin mRNA, accompanied by: (i) small, but significant, increase in serum urea after 24 h, (ii) 100% increase in serum creatinine at 24 h. A serum peak of inflammatory cytokines occurred after 5 days of reperfusion. Heart weight/body weight and heart weight/tibia length ratios increased after 12 and 15 days of reperfusion, respectively. Cardiac hypertrophy markers, B-type natriuretic peptide (BNP) and α-actin, left ventricle mass, cardiac wall thickness and myocyte width increased after 15 days of reperfusion, together with longer QTc and action potential duration. Cardiac TLRs, MyD88, HSP60 and HSP70 mRNA levels also increased. After 15 days of reperfusion, absence of TLRs prevented cardiac hypertrophy, as reflected by similar values of left ventricular cardiac mass and heart weight/body weight ratio compared to the transgenic Sham. Renal tissular injury also ameliorated in both knockout mice, as revealed by the comparison of their vimentin mRNA levels with those found in the WT on the same day after I/R. The I/R TLR2-/- group had TNF-α, IFN-γ and IL-1β levels similar to the non-I/R group, whereas the TLR4-/- group conserved the p-NF-κB/NF- κB ratio contrasting with that found in TLR2-/-. We conclude: (i) TLRs are involved in renal I/R-induced cardiac hypertrophy; (ii) absence of TLRs prevents I/R-induced cardiac hypertrophy, despite renal lesions seeming to evolve towards those of chronic disease; (iii) TLR2 and TLR4 selectively regulate the systemic inflammatory profile and NF- κB activation. PMID

  2. Activation and modulation of recombinantly expressed serotonin receptor type 3A by terpenes and pungent substances.

    PubMed

    Ziemba, Paul M; Schreiner, Benjamin S P; Flegel, Caroline; Herbrechter, Robin; Stark, Timo D; Hofmann, Thomas; Hatt, Hanns; Werner, Markus; Gisselmann, Günter

    2015-11-27

    Serotonin receptor type 3 (5-HT3 receptor) is a ligand-gated ion channel that is expressed in the central nervous system (CNS) as well as in the peripheral nervous system (PNS). The receptor plays an important role in regulating peristalsis of the gastrointestinal tract and in functions such as emesis, cognition and anxiety. Therefore, a variety of pharmacologically active substances target the 5-HT3 receptor to treat chemotherapy-induced nausea and vomiting. The 5-HT3 receptors are activated, antagonized, or modulated by a wide range of chemically different substances, such as 2-methyl-serotonin, phenylbiguanide, setrones, or cannabinoids. Whereas the action of all of these substances is well described, less is known about the effect of terpenoids or fragrances on 5-HT3A receptors. In this study, we screened a large number of natural odorous and pungent substances for their pharmacological action on recombinantly expressed human 5-HT3A receptors. The receptors were functionally expressed in Xenopus oocytes and characterized by electrophysiological recordings using the two-electrode voltage-clamp technique. A screening of two odorous mixes containing a total of 200 substances revealed that the monoterpenes, thymol and carvacrol, act as both weak partial agonists and positive modulators on the 5-HT3A receptor. In contrast, the most effective blockers were the terpenes, citronellol and geraniol, as well as the pungent substances gingerol, capsaicin and polygodial. In our study, we identified new modulators of 5-HT3A receptors out of the classes of monoterpenes and vanilloid substances that frequently occur in various plants. PMID:26456648

  3. Nature's knockout: the Mel1b receptor is not necessary for reproductive and circadian responses to melatonin in Siberian hamsters.

    PubMed

    Weaver, D R; Liu, C; Reppert, S M

    1996-11-01

    The pineal hormone melatonin regulates seasonal reproduction and influences the timing of circadian rhythms. The Mel1a and Mel1b receptors are the high-affinity melatonin receptors present in mammals. Unexpectedly, the Mel1b receptor gene of the Siberian hamster, Phodopus sungorus, cannot encode a functional receptor; two nonsense mutations are present within the coding region. Southern blot analysis indicates that this is a single copy gene. The Mel1b receptor gene is nonfunctional in outbred populations of P. sungorus and Phodopus campbelli. Siberian hamsters lacking a functional Mel1b receptor nevertheless show seasonal reproductive and circadian responses to melatonin, indicating that the Mel1b receptor is not necessary for these responses. These data support the hypothesis that the Mel1a receptor, which does encode a functional receptor in this species, mediates reproductive and circadian responses to melatonin.

  4. Mu Opioid Receptors on Primary Afferent Nav1.8 Neurons Contribute to Opiate-Induced Analgesia: Insight from Conditional Knockout Mice

    PubMed Central

    Karchewski, Laurie; Gardon, Olivier; Matifas, Audrey; Filliol, Dominique; Becker, Jérôme A. J.; Wood, John N.; Kieffer, Brigitte L.; Gaveriaux-Ruff, Claire

    2013-01-01

    Opiates are powerful drugs to treat severe pain, and act via mu opioid receptors distributed throughout the nervous system. Their clinical use is hampered by centrally-mediated adverse effects, including nausea or respiratory depression. Here we used a genetic approach to investigate the potential of peripheral mu opioid receptors as targets for pain treatment. We generated conditional knockout (cKO) mice in which mu opioid receptors are deleted specifically in primary afferent Nav1.8-positive neurons. Mutant animals were compared to controls for acute nociception, inflammatory pain, opiate-induced analgesia and constipation. There was a 76% decrease of mu receptor-positive neurons and a 60% reduction of mu-receptor mRNA in dorsal root ganglia of cKO mice. Mutant mice showed normal responses to heat, mechanical, visceral and chemical stimuli, as well as unchanged morphine antinociception and tolerance to antinociception in models of acute pain. Inflammatory pain developed similarly in cKO and controls mice after Complete Freund’s Adjuvant. In the inflammation model, however, opiate-induced (morphine, fentanyl and loperamide) analgesia was reduced in mutant mice as compared to controls, and abolished at low doses. Morphine-induced constipation remained intact in cKO mice. We therefore genetically demonstrate for the first time that mu opioid receptors partly mediate opiate analgesia at the level of Nav1.8-positive sensory neurons. In our study, this mechanism operates under conditions of inflammatory pain, but not nociception. Previous pharmacology suggests that peripheral opiates may be clinically useful, and our data further demonstrate that Nav1.8 neuron-associated mu opioid receptors are feasible targets to alleviate some forms of persistent pain. PMID:24069332

  5. A shutoff and exonuclease mutant of murine gammaherpesvirus-68 yields infectious virus and causes RNA loss in type I interferon receptor knockout cells.

    PubMed

    Sheridan, Victoria; Polychronopoulos, Louise; Dutia, Bernadette M; Ebrahimi, Bahram

    2014-05-01

    Significant loss of RNA followed by severely reduced cellular protein pool, a phenomenon termed host shutoff, is associated with a number of lytic virus infections and is a critical player in viral pathogenesis. Until recently, viral DNA exonucleases were associated only with processing of viral genomic DNA and its encapsidation. However, recent observations have identified host shutoff and exonuclease function for the highly conserved viral exonucleases in γ-herpesviruses, which include Kaposi's sarcoma-associated herpesvirus, Epstein-Barr virus and the mouse model murine gammaherpesvirus-68, also referred to as MHV-68. In this study, we show that although ablation of the MHV-68 exonuclease ORF37 caused a restrictive phenotype in WT IFN-α/β receptor-positive cells such as NIH 3T3, lack of ORF37 was tolerated in cells lacking the IFN-α/β receptor: the ORF37Stop virus was capable of forming infectious particles and caused loss of mRNA in IFN-α/β receptor knockout cells. Moreover, ORF37Stop virus was able to establish lytic infection in the lungs of mice lacking the IFN-α/β receptor. These observations provide evidence that lytic MHV-68 infection and subsequent loss of mRNA can take place independently of ORF37. Moreover, efficient growth of ORF37Stop virus also identifies a role for this family of viral nucleases in providing a window of opportunity for virus growth by overcoming type I IFN-dependent responses.

  6. Absence seizures in C3H/HeJ and knockout mice caused by mutation of the AMPA receptor subunit Gria4

    PubMed Central

    Beyer, Barbara; Deleuze, Charlotte; Letts, Verity A.; Mahaffey, Connie L.; Boumil, Rebecca M.; Lew, Timothy A.; Huguenard, John R.; Frankel, Wayne N.

    2008-01-01

    Absence epilepsy, characterized by spike–wave discharges (SWD) in the electroencephalogram, arises from aberrations within the circuitry of the cerebral cortex and thalamus that regulates awareness. The inbred mouse strain C3H/HeJ is prone to absence seizures, with a major susceptibility locus, spkw1, accounting for most of the phenotype. Here we find that spkw1 is associated with a hypomorphic retroviral-like insertion mutation in the Gria4 gene, encoding one of the four amino-3-hydroxy-5-methyl-4isoxazolepropionic acid (AMPA) receptor subunits in the brain. Consistent with this, Gria4 knockout mice also have frequent SWD and do not complement spkw1. In contrast, null mutants for the related gene Gria3 do not have SWD, and Gria3 loss actually lowers SWD of spkw1 homozygotes. Gria3 and Gria4 encode the predominant AMPA receptor subunits in the reticular thalamus, which is thought to play a central role in seizure genesis by inhibiting thalamic relay cells and promoting rebound burst firing responses. In Gria4 mutants, synaptic excitation of inhibitory reticular thalamic neurons is enhanced, with increased duration of synaptic responses—consistent with what might be expected from reduction of the kinetically faster subunit of AMPA receptors encoded by Gria4. These results demonstrate for the first time an essential role for Gria4 in the brain, and suggest that abnormal AMPA receptor-dependent synaptic activity can be involved in the network hypersynchrony that underlies absence seizures. PMID:18316356

  7. Effect of AVE 0991 angiotensin-(1-7) receptor agonist treatment on elemental and biomolecular content and distribution in atherosclerotic plaques of apoE-knockout mice

    NASA Astrophysics Data System (ADS)

    Kowalska, J.; Gajda, M.; Jawień, J.; Kwiatek, W. M.; Appel, K.; Dumas, P.

    2013-12-01

    Gene-targeted apolipoprotein E-knockout (apoE-KO) mice display early and highly progressive vascular lesions containing lipid deposits and they became a reliable animal model to study atherosclerosis. The aim of the present study was to investigate the effect of AVE 0991 angiotensin-(1-7) receptor agonist on the distribution of selected pro- and anti- inflammatory elements as well as biomolecules in atherosclerotic plaques of apoE-knockout mice. Synchrotron radiation-based X-ray fluorescence (micro-XRF) and Fourier Transform Infrared (micro-FTIR) microspectroscopies were applied. Two-month-old apoE-KO mice were fed for following four months diet supplemented with AVE 0991 (0.58 μmol/kg b.w. per day). Histological sections of ascending aortas were analyzed spectroscopically. The distribution of P, Ca, Fe and Zn were found to correspond with histological structure of the lesion. Significantly lower contents of P, Ca, Zn and significantly higher content of Fe were observed in animals treated with AVE 0991. Biomolecular analysis showed lower lipids saturation level and lower lipid to protein ratio in AVE 0991 treated group. Protein secondary structure was studied according to the composition of amide I band (1660 cm-1) and it demonstrated higher proportion of β-sheet structure as compared to α-helix in both studied groups.

  8. Knockout crickets for the study of learning and memory: Dopamine receptor Dop1 mediates aversive but not appetitive reinforcement in crickets.

    PubMed

    Awata, Hiroko; Watanabe, Takahito; Hamanaka, Yoshitaka; Mito, Taro; Noji, Sumihare; Mizunami, Makoto

    2015-01-01

    Elucidation of reinforcement mechanisms in associative learning is an important subject in neuroscience. In mammals, dopamine neurons are thought to play critical roles in mediating both appetitive and aversive reinforcement. Our pharmacological studies suggested that octopamine and dopamine neurons mediate reward and punishment, respectively, in crickets, but recent studies in fruit-flies concluded that dopamine neurons mediates both reward and punishment, via the type 1 dopamine receptor Dop1. To resolve the discrepancy between studies in different insect species, we produced Dop1 knockout crickets using the CRISPR/Cas9 system and found that they are defective in aversive learning with sodium chloride punishment but not appetitive learning with water or sucrose reward. The results suggest that dopamine and octopamine neurons mediate aversive and appetitive reinforcement, respectively, in crickets. We suggest unexpected diversity in neurotransmitters mediating appetitive reinforcement between crickets and fruit-flies, although the neurotransmitter mediating aversive reinforcement is conserved. This study demonstrates usefulness of the CRISPR/Cas9 system for producing knockout animals for the study of learning and memory. PMID:26521965

  9. Secretin receptor-knockout mice are resistant to high-fat diet-induced obesity and exhibit impaired intestinal lipid absorption.

    PubMed

    Sekar, Revathi; Chow, Billy K C

    2014-08-01

    Secretin, a classical gastrointestinal hormone released from S cells in response to acid and dietary lipid, regulates pleiotropic physiological functions, such as exocrine pancreatic secretion and gastric motility. Subsequent to recently proposed revisit on secretin's metabolic effects, we have confirmed lipolytic actions of secretin during starvation and discovered a hormone-sensitive lipase-mediated mechanistic pathway behind. In this study, a 12 wk high-fat diet (HFD) feeding to secretin receptor-knockout (SCTR(-/-)) mice and their wild-type (SCTR(+/+)) littermates revealed that, despite similar food intake, SCTR(-/-) mice gained significantly less weight (SCTR(+/+): 49.6±0.9 g; SCTR(-/-): 44.7±1.4 g; P<0.05) and exhibited lower body fat content. These SCTR(-/-) mice have corresponding alleviated HFD-associated hyperleptinemia and improved glucose/insulin tolerance. Further analyses indicate that SCTR(-/-) have impaired intestinal fatty acid absorption while having similar energy expenditure and locomotor activity. Reduced fat absorption in the intestine is further supported by lowered postprandial triglyceride concentrations in circulation in SCTR(-/-) mice. In jejunal cells, transcript and protein levels of a key fat absorption regulator, cluster of differentiation 36 (CD36), was reduced in knockout mice, while transcript of Cd36 and fatty-acid uptake in isolated enterocytes was stimulated by secretin. Based on our findings, a novel positive feedback pathway involving secretin and CD36 to enhance intestinal lipid absorption is being proposed.

  10. Knockout crickets for the study of learning and memory: Dopamine receptor Dop1 mediates aversive but not appetitive reinforcement in crickets.

    PubMed

    Awata, Hiroko; Watanabe, Takahito; Hamanaka, Yoshitaka; Mito, Taro; Noji, Sumihare; Mizunami, Makoto

    2015-11-02

    Elucidation of reinforcement mechanisms in associative learning is an important subject in neuroscience. In mammals, dopamine neurons are thought to play critical roles in mediating both appetitive and aversive reinforcement. Our pharmacological studies suggested that octopamine and dopamine neurons mediate reward and punishment, respectively, in crickets, but recent studies in fruit-flies concluded that dopamine neurons mediates both reward and punishment, via the type 1 dopamine receptor Dop1. To resolve the discrepancy between studies in different insect species, we produced Dop1 knockout crickets using the CRISPR/Cas9 system and found that they are defective in aversive learning with sodium chloride punishment but not appetitive learning with water or sucrose reward. The results suggest that dopamine and octopamine neurons mediate aversive and appetitive reinforcement, respectively, in crickets. We suggest unexpected diversity in neurotransmitters mediating appetitive reinforcement between crickets and fruit-flies, although the neurotransmitter mediating aversive reinforcement is conserved. This study demonstrates usefulness of the CRISPR/Cas9 system for producing knockout animals for the study of learning and memory.

  11. Knockout crickets for the study of learning and memory: Dopamine receptor Dop1 mediates aversive but not appetitive reinforcement in crickets

    PubMed Central

    Awata, Hiroko; Watanabe, Takahito; Hamanaka, Yoshitaka; Mito, Taro; Noji, Sumihare; Mizunami, Makoto

    2015-01-01

    Elucidation of reinforcement mechanisms in associative learning is an important subject in neuroscience. In mammals, dopamine neurons are thought to play critical roles in mediating both appetitive and aversive reinforcement. Our pharmacological studies suggested that octopamine and dopamine neurons mediate reward and punishment, respectively, in crickets, but recent studies in fruit-flies concluded that dopamine neurons mediates both reward and punishment, via the type 1 dopamine receptor Dop1. To resolve the discrepancy between studies in different insect species, we produced Dop1 knockout crickets using the CRISPR/Cas9 system and found that they are defective in aversive learning with sodium chloride punishment but not appetitive learning with water or sucrose reward. The results suggest that dopamine and octopamine neurons mediate aversive and appetitive reinforcement, respectively, in crickets. We suggest unexpected diversity in neurotransmitters mediating appetitive reinforcement between crickets and fruit-flies, although the neurotransmitter mediating aversive reinforcement is conserved. This study demonstrates usefulness of the CRISPR/Cas9 system for producing knockout animals for the study of learning and memory. PMID:26521965

  12. Long-lived growth hormone receptor knockout mice show a delay in age-related changes of body composition and bone characteristics.

    PubMed

    Bonkowski, Michael S; Pamenter, Richard W; Rocha, Juliana S; Masternak, Michal M; Panici, Jacob A; Bartke, Andrzej

    2006-06-01

    There is conflicting information on the physiological role of growth hormone (GH) in the control of aging. This study reports dual-energy x-ray absorptiometry (DXA) measurements of body composition and bone characteristics in young, adult, and aged long-lived GH receptor knockout (GHR-KO) and normal mice to determine the effects of GH resistance during aging. Compared to controls, GHR-KO mice showed an increased percentage of body fat. GHR-KO mice have reduced total-body bone mineral density (BMD), bone mineral content, and bone area, but these parameters increased with age. In addition, GHR-KO mice have decreased femur length, femur BMD, and lower lumbar BMD compared to controls in all age groups. These parameters also continued to increase with age. Our results indicate that GH resistance alters body composition, bone growth, and bone maintenance during aging in GHR-KO mice.

  13. Rescue of a Recombinant Machupo Virus from Cloned cDNAs and In Vivo Characterization in Interferon (αβ/γ) Receptor Double Knockout Mice

    PubMed Central

    Patterson, Michael; Seregin, Alexey; Huang, Cheng; Kolokoltsova, Olga; Smith, Jennifer; Miller, Milagros; Smith, Jeanon; Yun, Nadezhda; Poussard, Allison; Grant, Ashley; Tigabu, Bersabeh; Walker, Aida

    2014-01-01

    Machupo virus (MACV) is the etiological agent of Bolivian hemorrhagic fever (BHF), a reemerging and neglected tropical disease associated with high mortality. The prototypical strain of MACV, Carvallo, was isolated from a human patient in 1963, but minimal in vitro and in vivo characterization has been reported. To this end, we utilized reverse genetics to rescue a pathogenic MACV from cloned cDNAs. The recombinant MACV (rMACV) had in vitro growth properties similar to those of the parental MACV. Both viruses caused similar disease development in alpha/beta and gamma interferon receptor knockout mice, including neurological disease development and high mortality. In addition, we have identified a novel murine model with mortality and neurological disease similar to BHF disease reported in humans and nonhuman primates. PMID:24284323

  14. Rescue of a recombinant Machupo virus from cloned cDNAs and in vivo characterization in interferon (αβ/γ) receptor double knockout mice.

    PubMed

    Patterson, Michael; Seregin, Alexey; Huang, Cheng; Kolokoltsova, Olga; Smith, Jennifer; Miller, Milagros; Smith, Jeanon; Yun, Nadezhda; Poussard, Allison; Grant, Ashley; Tigabu, Bersabeh; Walker, Aida; Paessler, Slobodan

    2014-02-01

    Machupo virus (MACV) is the etiological agent of Bolivian hemorrhagic fever (BHF), a reemerging and neglected tropical disease associated with high mortality. The prototypical strain of MACV, Carvallo, was isolated from a human patient in 1963, but minimal in vitro and in vivo characterization has been reported. To this end, we utilized reverse genetics to rescue a pathogenic MACV from cloned cDNAs. The recombinant MACV (rMACV) had in vitro growth properties similar to those of the parental MACV. Both viruses caused similar disease development in alpha/beta and gamma interferon receptor knockout mice, including neurological disease development and high mortality. In addition, we have identified a novel murine model with mortality and neurological disease similar to BHF disease reported in humans and nonhuman primates.

  15. Evidence from knockout mice for distinct implications of neuropeptide-Y Y2 and Y4 receptors in the circadian control of locomotion, exploration, water and food intake.

    PubMed

    Edelsbrunner, M E; Painsipp, E; Herzog, H; Holzer, P

    2009-12-01

    Members of the neuropeptide-Y (NPY) family acting via Y2 and/or Y4 receptors have been proposed to participate in the control of ingestive behaviour and energy homeostasis. Since these processes vary between day and night, we explored the circadian patterns of locomotor, exploratory and ingestive behaviour in mice with disrupted genes for Y2 (Y2-/-) or Y4 (Y4-/-) receptors. To this end, the LabMaster system was used and its utility for the analysis of changes in circadian activity and ingestion caused by gene knockout evaluated. Female animals, aged 27weeks on average, were housed singly in cages fitted with sensors for water and food intake and two infrared frames for recording ambulation and rearing under a 12h light/dark cycle for 4days. Relative to WT animals, diurnal locomotion, exploration, drinking and feeding were reduced, whereas nocturnal locomotion was enhanced in Y2-/- mice. In contrast, Y4-/- mice moved more but ate and drank less during the photophase, while they ate more and explored less during the scotophase. Both Y2-/- and Y4-/- mice weighed more than WT mice. These findings attest to a differential role of Y2 and Y4 receptor signalling in the circadian control of behaviours that balance energy intake and energy expenditure. These phenotypic traits can be sensitively and continuously recorded by the LabMaster system.

  16. Immunohistochemical distribution of somatostatin and somatostatin receptor subtypes (SSTR1-5) in hypothalamus of ApoD knockout mice brain.

    PubMed

    Kumar, Ujendra

    2012-11-01

    In the present study, the expression of somatostatin (SST) and somatostatin receptor subtypes (SSTR1-5) was determined in the hypothalamus of wild-type (wt) and apolipoprotein D knockout (ApoD(-/-)) mice brain. SST-like immunoreactivity, while comparable in most regions of hypothalamus, diminished significantly in arcuate nucleus of ApoD(-/-) mice. SSTR1 strongly localized in all major hypothalamic nuclei as well as in the median eminence and ependyma of the third ventricle of wt mice brain. SSTR1-like immunoreactivity increases in hypothalamus except in paraventricular nucleus of ApoD(-/-) mice. SSTR2 was well expressed in most of the hypothalamic regions whereas it decreases significantly in ventromedial and arcuate nucleus of ApoD(-/-) mice. SSTR3 and SSTR4-like immunoreactivity increases in ApoD(-/-) mice in all major nuclei of hypothalamus, median eminence, and ependymal cells of third ventricle. SSTR5 is well expressed in ventromedial and arcuate nucleus whereas weakly expressed in paraventricular nucleus. In comparison to wt, ApoD(-/-) mice exhibit increased SSTR5-like immunoreactivity in paraventricular nuclei and decreased receptor expression in ventromedial hypothalamus and arcuate nucleus. In conclusion, the changes in hypothalamus of ApoD(-/-) mice may indicate potential role of ApoD in regulation of endocrine functions of somatostatin in a receptor-dependent manner.

  17. Mitochondrial gene expression and increased oxidative metabolism: role in increased lifespan of fat-specific insulin receptor knock-out mice.

    PubMed

    Katic, Masa; Kennedy, Adam R; Leykin, Igor; Norris, Andrew; McGettrick, Aileen; Gesta, Stephane; Russell, Steven J; Bluher, Matthias; Maratos-Flier, Eleftheria; Kahn, C Ronald

    2007-12-01

    Caloric restriction, leanness and decreased activity of insulin/insulin-like growth factor 1 (IGF-1) receptor signaling are associated with increased longevity in a wide range of organisms from Caenorhabditis elegans to humans. Fat-specific insulin receptor knock-out (FIRKO) mice represent an interesting dichotomy, with leanness and increased lifespan, despite normal or increased food intake. To determine the mechanisms by which a lack of insulin signaling in adipose tissue might exert this effect, we performed physiological and gene expression studies in FIRKO and control mice as they aged. At the whole body level, FIRKO mice demonstrated an increase in basal metabolic rate and respiratory exchange ratio. Analysis of gene expression in white adipose tissue (WAT) of FIRKO mice from 6 to 36 months of age revealed persistently high expression of the nuclear-encoded mitochondrial genes involved in glycolysis, tricarboxylic acid cycle, beta-oxidation and oxidative phosphorylation as compared to expression of the same genes in WAT from controls that showed a tendency to decline in expression with age. These changes in gene expression were correlated with increased cytochrome c and cytochrome c oxidase subunit IV at the protein level, increased citrate synthase activity, increased expression of peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha) and PGC-1beta, and an increase in mitochondrial DNA in WAT of FIRKO mice. Together, these data suggest that maintenance of mitochondrial activity and metabolic rates in adipose tissue may be important contributors to the increased lifespan of the FIRKO mouse.

  18. DECREASED EXPRESSION LEVEL OF APOPTOSIS-RELATED GENES AND/OR PROTEINS IN SKELETAL MUSCLES, BUT NOT IN HEARTS, OF GROWTH HORMONE RECEPTOR KNOCKOUT MICE

    PubMed Central

    Gesing, Adam; Masternak, Michal M.; Wang, Feiya; Lewinski, Andrzej; Karbownik-Lewinska, Malgorzata; Bartke, Andrzej

    2013-01-01

    The long-lived growth hormone (GH) receptor knockout (GHRKO; KO) mice are GH resistant due to targeted disruption of the GH receptor (Ghr) gene. Apoptosis is a physiological process in which cells play an active role in their own death and is a normal component of the development and health of multicellular organisms. Aging is associated with the progressive loss of strength of skeletal and heart muscles. Calorie restriction (CR) is a well known experimental model to delay aging and increase lifespan. The aim of the study was to examine the expression of the following apoptosis-related genes: caspase-3, caspase-9, caspase-8, bax, bcl-2, Smac/DIABLO, p53 and cytochrome c1 (cyc1) in the skeletal muscles and hearts of female normal and GHRKO mice, fed ad libitum or subjected to 40% CR for 6 months, starting at 2 months of age. Moreover, skeletal muscle caspase-3, caspase-9, caspase-8, bax, bcl-2, Smac/DIABLO, Apaf-1, bad, phospho-bad (pbad), phospho-p53 (pp53) and cytochrome c (cyc) protein expression levels were assessed. Results Expression of caspase-3, caspase-9, bax and Smac/DIABLO genes and proteins was decreased in GHRKO’s skeletal muscles. The Apaf-1 protein expression also was diminished in this tissue. In contrast, bcl-2 and pbad protein levels were increased in skeletal muscles in knockouts. No changes were demonstrated for the examined genes expression in GHRKO’s hearts except for the increased level of cyc1 mRNA. CR did not alter the expression of the examined genes and proteins in skeletal muscles of knockouts vs. normal (N) mice. In heart homogenates, CR increased caspase-3 mRNA level as compared to ad libitum (AL) mice. Conclusion decreased expression of certain pro-apoptotic genes and/or proteins may constitute the potential mechanism of prolonged longevity in GHRKO mice, protecting these animals from aging; this potential beneficial mechanism is not affected by calorie restriction. PMID:21321312

  19. Genomic Profiling of Tumor Necrosis Factor Alpha (TNF-α) Receptor and Interleukin-1 Receptor Knockout Mice Reveals a Link between TNF-α Signaling and Increased Severity of 1918 Pandemic Influenza Virus Infection▿ †

    PubMed Central

    Belisle, Sarah E.; Tisoncik, Jennifer R.; Korth, Marcus J.; Carter, Victoria S.; Proll, Sean C.; Swayne, David E.; Pantin-Jackwood, Mary; Tumpey, Terrence M.; Katze, Michael G.

    2010-01-01

    The influenza pandemic of 1918 to 1919 was one of the worst global pandemics in recent history. The highly pathogenic nature of the 1918 virus is thought to be mediated in part by a dysregulation of the host response, including an exacerbated proinflammatory cytokine response. In the present study, we compared the host transcriptional response to infection with the reconstructed 1918 virus in wild-type, tumor necrosis factor (TNF) receptor-1 knockout (TNFRKO), and interleukin-1 (IL-1) receptor-1 knockout (IL1RKO) mice as a means of further understanding the role of proinflammatory cytokine signaling during the acute response to infection. Despite reported redundancy in the functions of IL-1β and TNF-α, we observed that reducing the signaling capacity of each of these molecules by genetic disruption of their key receptor genes had very different effects on the host response to infection. In TNFRKO mice, we found delayed or decreased expression of genes associated with antiviral and innate immune signaling, complement, coagulation, and negative acute-phase response. In contrast, in IL1RKO mice numerous genes were differentially expressed at 1 day postinoculation, including an increase in the expression of genes that contribute to dendritic and natural killer cell processes and cellular movement, and gene expression profiles remained relatively constant at later time points. We also observed a compensatory increase in TNF-α expression in virus-infected IL1RKO mice. Our data suggest that signaling through the IL-1 receptor is protective, whereas signaling through the TNF-α receptor increases the severity of 1918 virus infection. These findings suggest that manipulation of these pathways may have therapeutic benefit. PMID:20926563

  20. Genomic profiling of tumor necrosis factor alpha (TNF-alpha) receptor and interleukin-1 receptor knockout mice reveals a link between TNF-alpha signaling and increased severity of 1918 pandemic influenza virus infection.

    PubMed

    Belisle, Sarah E; Tisoncik, Jennifer R; Korth, Marcus J; Carter, Victoria S; Proll, Sean C; Swayne, David E; Pantin-Jackwood, Mary; Tumpey, Terrence M; Katze, Michael G

    2010-12-01

    The influenza pandemic of 1918 to 1919 was one of the worst global pandemics in recent history. The highly pathogenic nature of the 1918 virus is thought to be mediated in part by a dysregulation of the host response, including an exacerbated proinflammatory cytokine response. In the present study, we compared the host transcriptional response to infection with the reconstructed 1918 virus in wild-type, tumor necrosis factor (TNF) receptor-1 knockout (TNFRKO), and interleukin-1 (IL-1) receptor-1 knockout (IL1RKO) mice as a means of further understanding the role of proinflammatory cytokine signaling during the acute response to infection. Despite reported redundancy in the functions of IL-1β and TNF-α, we observed that reducing the signaling capacity of each of these molecules by genetic disruption of their key receptor genes had very different effects on the host response to infection. In TNFRKO mice, we found delayed or decreased expression of genes associated with antiviral and innate immune signaling, complement, coagulation, and negative acute-phase response. In contrast, in IL1RKO mice numerous genes were differentially expressed at 1 day postinoculation, including an increase in the expression of genes that contribute to dendritic and natural killer cell processes and cellular movement, and gene expression profiles remained relatively constant at later time points. We also observed a compensatory increase in TNF-α expression in virus-infected IL1RKO mice. Our data suggest that signaling through the IL-1 receptor is protective, whereas signaling through the TNF-α receptor increases the severity of 1918 virus infection. These findings suggest that manipulation of these pathways may have therapeutic benefit.

  1. Relaxin-3 receptor (Rxfp3) gene knockout mice display reduced running wheel activity: implications for role of relaxin-3/RXFP3 signalling in sustained arousal.

    PubMed

    Hosken, Ihaia T; Sutton, Steven W; Smith, Craig M; Gundlach, Andrew L

    2015-02-01

    Anatomical and pharmacological evidence suggests the neuropeptide, relaxin-3, is the preferred endogenous ligand for the relaxin family peptide-3 receptor (RXFP3) and suggests a number of putative stress- and arousal-related roles for RXFP3 signalling. However, in vitro and in vivo evidence demonstrates exogenous relaxin-3 can activate other relaxin peptide family receptors, and the role of relaxin-3/RXFP3 signalling in specific brain circuits and associated behaviours in mice is not well described. In this study, we characterised the behaviour of cohorts of male and female Rxfp3 gene knockout (KO) mice (C57/B6J(RXFP3TM1/DGen)), relative to wild-type (WT) littermates to determine if this receptor KO strain has a similar phenotype to its ligand KO equivalent. Rxfp3 KO mice displayed similar performance to WT littermates in several acute behavioural paradigms designed to gauge motor coordination (rotarod test), spatial memory (Y-maze), depressive-like behaviour (repeat forced-swim test) and sensorimotor gating (prepulse inhibition of acoustic startle). Notably however, male and female Rxfp3 KO mice displayed robust and consistent (dark phase) hypoactivity on voluntary home-cage running wheels (∼20-60% less activity/h), and a small but significant decrease in anxiety-like behavioural traits in the elevated plus maze and light/dark box paradigms. Importantly, this phenotype is near identical to that observed in two independent lines of relaxin-3 KO mice, suggesting these phenotypes are due to the elimination of ligand or receptor and RXFP3-linked signalling. Furthermore, this behavioural characterisation of Rxfp3 KO mice identifies them as a useful experimental model for studying RXFP3-linked signalling and assessing the selectivity and/or potential off-target actions of RXFP3 agonists and antagonists, which could lead to an improved understanding of dysfunctional arousal in mental health disorders, including depression, anxiety, insomnia and neurodegenerative

  2. Behavioural and biochemical responses to morphine associated with its motivational properties are altered in adenosine A2A receptor knockout mice

    PubMed Central

    Castañé, A; Wells, L; Soria, G; Hourani, S; Ledent, C; Kitchen, I; Opacka-Juffry, J; Maldonado, R; Valverde, O

    2008-01-01

    Background and purpose: The purinergic system through the A2A adenosine receptor regulates addiction induced by different drugs of abuse. The aim of the present study was to investigate the specific role of A2A adenosine receptors (A2ARs) in the behavioural and neurochemical responses to morphine associated with its motivational properties. Experimental approach: Mice lacking A2ARs (A2A knockout (KO) mice) and wild-type littermates were used to evaluate behavioural responses induced by morphine. Antinociception was assessed using the tail-immersion and the hot-plate tests. Place-conditioning paradigms were used to evaluate the rewarding effects of morphine and the dysphoric responses of morphine withdrawal. Microdialysis studies were carried out to evaluate changes in the extracellular levels of dopamine in the nucleus accumbens of A2A KO mice after morphine administration. Key results: The acute administration of morphine induced a similar enhancement of locomotor activity and antinociceptive responses in both genotypes. However, the rewarding effects induced by morphine were completely blocked in A2A KO mice. Also, naloxone did not induce place aversion in animals lacking the A2ARs. Conclusions and implications: Our findings demonstrate that the rewarding and aversive effects associated with morphine abstinence were abolished in A2A KO mice, supporting a differential role of the A2A adenosine receptor in the somatic and motivational effects of morphine addiction. This study provides evidence for the role of A2ARs as general modulators of the motivational properties of drugs of abuse. Pharmacological manipulation of these receptors may represent a new target in the management of drug addiction. PMID:18660831

  3. The low density lipoprotein receptor-related protein 1: Unique tissue-specific functions revealed by selective gene knockout studies

    PubMed Central

    Lillis, Anna P.; Van Duyn, Lauren B.; Murphy-Ullrich, Joanne E.; Strickland, Dudley K.

    2008-01-01

    The low-density lipoprotein (LDL) receptor-related protein (originally called LRP, but now referred to as LRP1) is a large endocytic receptor that is widely expressed in several tissues. LRP1 is a member of the LDL receptor family that plays diverse roles in various biological processes including lipoprotein metabolism, degradation of proteases, activation of lysosomal enzymes and cellular entry of bacterial toxins and viruses. Deletion of the LRP1 gene leads to lethality in mice, revealing a critical, but as of yet, undefined role in development. Tissue-specific gene deletion studies reveal an important contribution of LRP1 in the vasculature, central nervous system, in macrophages and in adipocytes. Three important properties of LRP1 dictate its diverse role in physiology: first, its ability to recognize more than thirty distinct ligands; second, its ability to bind a large number of cytoplasmic adaptor proteins via determinants located on its cytoplasmic domain in a phosphorylation-specific manner; and third, its ability to associate with and modulate the activity of other transmembrane receptors such as integrins and receptor tyrosine kinases. PMID:18626063

  4. High Affinity Dopamine D3 Receptor (D3R)-Selective Antagonists Attenuate Heroin Self-Administration in Wild-Type but not D3R Knockout Mice

    PubMed Central

    2015-01-01

    The dopamine D3 receptor (D3R) is a promising target for the development of pharmacotherapeutics to treat substance use disorders. Several D3R-selective antagonists are effective in animal models of drug abuse, especially in models of relapse. Nevertheless, poor bioavailability, metabolic instability, and/or predicted toxicity have impeded success in translating these drug candidates to clinical use. Herein, we report a series of D3R-selective 4-phenylpiperazines with improved metabolic stability. A subset of these compounds was evaluated for D3R functional efficacy and off-target binding at selected 5-HT receptor subtypes, where significant overlap in SAR with D3R has been observed. Several high affinity D3R antagonists, including compounds 16 (Ki = 0.12 nM) and 32 (Ki = 0.35 nM), showed improved metabolic stability compared to the parent compound, PG648 (6). Notably, 16 and the classic D3R antagonist SB277011A (2) were effective in reducing self-administration of heroin in wild-type but not D3R knockout mice. PMID:26203768

  5. Genetic knockout of the α7 nicotinic acetylcholine receptor gene alters hippocampal long-term potentiation in a background strain-dependent manner.

    PubMed

    Freund, Ronald K; Graw, Sharon; Choo, Kevin S; Stevens, Karen E; Leonard, Sherry; Dell'Acqua, Mark L

    2016-08-01

    Reduced α7 nicotinic acetylcholine receptor (nAChR) function is linked to impaired hippocampal-dependent sensory processing and learning and memory in schizophrenia. While knockout of the Chrna7 gene encoding the α7nAChR on a C57/Bl6 background results in changes in cognitive measures, prior studies found little impact on hippocampal synaptic plasticity in these mice. However, schizophrenia is a multi-genic disorder where complex interactions between specific genetic mutations and overall genetic background may play a prominent role in determining phenotypic penetrance. Thus, we compared the consequences of knocking out the α7nAChR on synaptic plasticity in C57/Bl6 and C3H mice, which differ in their basal α7nAChR expression levels. Homozygous α7 deletion in C3H mice, which normally express higher α7nAChR levels, resulted in impaired long-term potentiation (LTP) at hippocampal CA1 synapses, while C3H α7 heterozygous mice maintained robust LTP. In contrast, homozygous α7 deletion in C57 mice, which normally express lower α7nAChR levels, did not alter LTP, as had been previously reported for this strain. Thus, the threshold of Chrna7 expression required for LTP may be different in the two strains. Measurements of auditory gating, a hippocampal-dependent behavioral paradigm used to identify schizophrenia-associated sensory processing deficits, was abnormal in C3H α7 knockout mice confirming that auditory gating also requires α7nAChR expression. Our studies highlight the importance of genetic background on the regulation of synaptic plasticity and could be relevant for understanding genetic and cognitive heterogeneity in human studies of α7nAChR dysfunction in mental disorders.

  6. Peripheral Benzodiazepine Receptor/Translocator Protein Global Knock-out Mice Are Viable with No Effects on Steroid Hormone Biosynthesis*♦

    PubMed Central

    Tu, Lan N.; Morohaku, Kanako; Manna, Pulak R.; Pelton, Susanne H.; Butler, W. Ronald; Stocco, Douglas M.; Selvaraj, Vimal

    2014-01-01

    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. PMID:24936060

  7. Growth hormone secretagogue receptor (GHS-R1a) knockout mice exhibit improved spatial memory and deficits in contextual memory.

    PubMed

    Albarran-Zeckler, Rosie G; Brantley, Alicia Faruzzi; Smith, Roy G

    2012-06-15

    Although the hormone ghrelin is best known for its stimulatory effect on appetite and regulation of growth hormone release, it is also reported to have beneficial effects on learning and memory formation in mice. Nevertheless, controversy exists about whether endogenous ghrelin acts on its receptors in extra-hypothalamic areas of the brain. The ghrelin receptor (GHS-R1a) is co-expressed in neurons that express dopamine receptor type-1 (DRD1a) and type-2 (DRD2), and we have shown that a subset of GHS-R1a, which are not occupied by the agonist (apo-GHSR1a), heterodimerize with these two receptors to regulate dopamine signaling in vitro and in vivo. To determine the consequences of ghsr ablation on brain function, congenic ghsr -/- mice on the C57BL6/J background were subjected to a battery of behavioral tests. We show that the ghsr -/- mice exhibit normal balance, movement, coordination, and pain sensation, outperform ghsr +/+ mice in the Morris water maze, but show deficits in contextual fear conditioning.

  8. Insulin and insulin-like growth factor-I (IGF-I) receptor phosphorylation in µ-calpain knockout mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Numerous cellular processes are controlled by insulin and IGF-I signaling pathways. Due to previous work in our laboratories, we hypothesized that insulin (IR) and type 1 IGF-I (IGF-IR) receptor signaling is decreased due to increased protein tyrosine phosphatase 1B (PTP1B) activity. C57BL/6J mice...

  9. Effects of female pheromones on gonadotropin-releasing hormone gene expression and luteinizing hormone release in male wild-type and oestrogen receptor-alpha knockout mice.

    PubMed

    Gore, A C; Wersinger, S R; Rissman, E F

    2000-12-01

    Pheromones are an important class of environmental cues that affect the hypothalamic-pituitary-gonadal axis in a variety of vertebrate species, including humans. When male mice contact female-soiled bedding, or urine, they display a reflexive luteinizing hormone (LH) surge within 30 min. Aside from the requirement that males have gonads to show this response, the physiological mechanisms that underlie this pituitary response are unknown. In this experiment, we asked if female pheromones acted at the level of gonadotropin-releasing hormone (GnRH) gene expression to affect this hormone response. In addition, we also examined the contribution of one of the oestrogen receptors (ERalpha) by studying this neuroendocrine reflex in wild-type and oestrogen receptor-alpha knockout (ERalphaKO) males. Both ERalphaKO and wild-type males showed the expected LH surge, 45 and 90 min after contact with female pheromones. Males housed in clean bedding or bedding soiled by another adult male did not display the LH elevation. Interestingly, this dramatic change in LH concentrations was not accompanied by any alterations in GnRH mRNA expression or levels of primary transcript in the preoptic area-anterior hypothalamus. The one exception to this was a significant increase in GnRH mRNA expression in tissue collected from wild-type males exposed to bedding from another male. This is particularly intriguing since LH was not elevated in these males. These data replicate and extend our previous finding that ERalphaKO males do exhibit an LH surge in response to female pheromones. Thus, this neuroendocrine response is regulated by a steroid receptor other than ERalpha and does not require alterations in GnRH mRNA expression.

  10. The role of group II metabotropic glutamate receptors in cognition and anxiety: comparative studies in GRM2(-/-), GRM3(-/-) and GRM2/3(-/-) knockout mice.

    PubMed

    De Filippis, Bianca; Lyon, Louisa; Taylor, Amy; Lane, Tracy; Burnet, Philip W J; Harrison, Paul J; Bannerman, David M

    2015-02-01

    Group II metabotropic glutamate receptors (mGlu2 and mGlu3, encoded by GRM2 and GRM3) have been implicated in both cognitive and emotional processes, although their precise role remains to be established. Studies with knockout (KO) mice provide an important approach for investigating the role of specific receptor genes in behaviour. In the present series of experiments we extended our prior characterisation of GRM2/3(-/-) double KO mice and, in complementary experiments, investigated the behavioural phenotype of single GRM2(-/-) and GRM3(-/-) mice. We found no consistent effect on anxiety in either the double or single KO mice. The lack of an anxiety phenotype in any of the lines contrasts with the clear anxiolytic effects of mGlu2/3 ligands. Motor co-ordination was impaired in GRM2/3(-/-) mice, but spared in single GRM2(-/-) and GRM3(-/-) mice. Spatial working memory (rewarded alternation) testing on the elevated T-maze revealed a deficit in GRM2(-/-) mice throughout testing, whereas GRM3(-/-) mice exhibited a biphasic effect (initially impaired, but performing better than controls by the end of training). A biphasic effect on activity levels was seen for the GRM2(-/-) mice. Overall, the phenotype in both GRM2(-/-) and GRM3(-/-) mice was less pronounced - if present at all - compared to GRM2/3(-/-) mice, across the range of task domains. This is consistent with possible redundancy of function and/or compensation in the single KO lines. Results are discussed with reference to a possible role for group II metabotropic glutamate receptors at the interface between arousal and behavioural performance, according to an inverted U-shaped function. PMID:25158312

  11. Cognitive and socio-emotional deficits in platelet-derived growth factor receptor-β gene knockout mice.

    PubMed

    Nguyen, Phuong Thi Hong; Nakamura, Tomoya; Hori, Etsuro; Urakawa, Susumu; Uwano, Teruko; Zhao, Juanjuan; Li, Ruixi; Bac, Nguyen Duy; Hamashima, Takeru; Ishii, Yoko; Matsushima, Takako; Ono, Taketoshi; Sasahara, Masakiyo; Nishijo, Hisao

    2011-01-01

    Platelet-derived growth factor (PDGF) is a potent mitogen. Extensive in vivo studies of PDGF and its receptor (PDGFR) genes have reported that PDGF plays an important role in embryogenesis and development of the central nervous system (CNS). Furthermore, PDGF and the β subunit of the PDGF receptor (PDGFR-β) have been reported to be associated with schizophrenia and autism. However, no study has reported on the effects of PDGF deletion on mice behavior. Here we generated novel mutant mice (PDGFR-β KO) in which PDGFR-β was conditionally deleted in CNS neurons using the Cre/loxP system. Mice without the Cre transgene but with floxed PDGFR-β were used as controls. Both groups of mice reached adulthood without any apparent anatomical defects. These mice were further examined by conducting several behavioral tests for spatial memory, social interaction, conditioning, prepulse inhibition, and forced swimming. The test results indicated that the PDGFR-β KO mice show deficits in all of these areas. Furthermore, an immunohistochemical study of the PDGFR-β KO mice brain indicated that the number of parvalbumin (calcium-binding protein)-positive (i.e., putatively γ-aminobutyric acid-ergic) neurons was low in the amygdala, hippocampus, and medial prefrontal cortex. Neurophysiological studies indicated that sensory-evoked gamma oscillation was low in the PDGFR-β KO mice, consistent with the observed reduction in the number of parvalbumin-positive neurons. These results suggest that PDGFR-β plays an important role in cognitive and socioemotional functions, and that deficits in this receptor may partly underlie the cognitive and socioemotional deficits observed in schizophrenic and autistic patients. PMID:21437241

  12. Pharmacological enhancement of mGlu5 receptors rescues behavioral deficits in SHANK3 knock-out mice

    PubMed Central

    Vicidomini, Cinzia; Ponzoni, Luisa; Lim, Dmitry; Schmeisser, Michael; Reim, Dominik; Morello, Noemi; Orelanna, Daniel; Tozzi, Alessandro; Durante, Valentina; Scalmani, Paolo; Mantegazza, Massimo; Genazzani, Armando A.; Giustetto, Maurizio; Sala, Mariaelvina; Calabresi, Paolo; Boeckers, Tobias M.; Sala, Carlo; Verpelli, Chiara

    2016-01-01

    SHANK3 (also called PROSAP2) genetic haploinsufficiency is thought to be the major cause of neuropsychiatric symptoms in Phelan-McDermid syndrome (PMS). PMS is a rare genetic disorder that causes a severe form of intellectual disability (ID), expressive language delays and other autistic features. Furthermore, a significant number of SHANK3 mutations have been identified in patients with Autism Spectrum disorders ASD, and SHANK3 truncating mutations are associated with moderate to profound ID. The Shank3 protein is a scaffold protein that is located in the postsynaptic density (PSD) of excitatory synapses and is crucial for synapse development and plasticity. In this study, we investigated the molecular mechanisms associated with the ASD-like behaviors observed in Shank3Δ11-/- mice in which exon 11 has been deleted. Our results indicate that Shank3 is essential to mediating mGlu5 receptor signaling by recruiting Homer1b/c to the PSD, specifically in the striatum and cortex. Moreover, augmenting mGlu5 receptor activity by administering 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) ameliorated the functional and behavioral defects that were observed in Shank3Δ11-/- mice, suggesting that pharmaceutical treatments that increase mGlu5 activity may represent a new approach for treating patients that are affected by PMS and SHANK3 mutations. PMID:27021819

  13. Gene knockout of nuclear progesterone receptor provides insights into the regulation of ovulation by LH signaling in zebrafish

    PubMed Central

    Tang, Haipei; Liu, Yun; Li, Jianzhen; Yin, Yike; Li, Gaofei; Chen, Yu; Li, Shuisheng; Zhang, Yong; Lin, Haoran; Liu, Xiaochun; Cheng, Christopher H. K.

    2016-01-01

    It is well established that the luteinizing hormone surge triggers ovulation, a dynamic process leading to the release of the mature oocyte from the ovarian follicle. But how this process controlled by LH signaling remains largely unknown in non-mammalian species. In this study, we investigated the roles of nuclear progesterone receptor (npr) in LH-induced ovulation. Our results indicate that the nuclear progesterone receptor serves as an important mediator of LH action on ovulation. This conclusion is based on the following results: (1) the expression level of npr peaks at the full-grown stage of the follicles; (2) the expression of npr is stimulated by LH signaling in vitro and in vivo; and (3) the npr null females are infertile due to ovulation defects. Moreover, we further show that LH signaling could induce ptger4b expression in an npr-dependent manner, and blockage of Ptger4b could also block hCG-induced ovulation. Collectively, our results not only demonstrate that npr serves an indispensable role in mediating the action of LH on ovulation in zebrafish, but also provide insights into the molecular mechanisms of the regulation of ovulation in fish. PMID:27333837

  14. Generation of Interleukin-2 Receptor Gamma Gene Knockout Pigs from Somatic Cells Genetically Modified by Zinc Finger Nuclease-Encoding mRNA

    PubMed Central

    Watanabe, Masahito; Nakano, Kazuaki; Matsunari, Hitomi; Matsuda, Taisuke; Maehara, Miki; Kanai, Takahiro; Kobayashi, Mirina; Matsumura, Yukina; Sakai, Rieko; Kuramoto, Momoko; Hayashida, Gota; Asano, Yoshinori; Takayanagi, Shuko; Arai, Yoshikazu; Umeyama, Kazuhiro; Nagaya, Masaki; Hanazono, Yutaka; Nagashima, Hiroshi

    2013-01-01

    Zinc finger nuclease (ZFN) is a powerful tool for genome editing. ZFN-encoding plasmid DNA expression systems have been recently employed for the generation of gene knockout (KO) pigs, although one major limitation of this technology is the use of potentially harmful genome-integrating plasmid DNAs. Here we describe a simple, non-integrating strategy for generating KO pigs using ZFN-encoding mRNA. The interleukin-2 receptor gamma (IL2RG) gene was knocked out in porcine fetal fibroblasts using ZFN-encoding mRNAs, and IL2RG KO pigs were subsequently generated using these KO cells through somatic cell nuclear transfer (SCNT). The resulting IL2RG KO pigs completely lacked a thymus and were deficient in T and NK cells, similar to human X-linked SCID patients. Our findings demonstrate that the combination of ZFN-encoding mRNAs and SCNT provides a simple robust method for producing KO pigs without genomic integration. PMID:24130776

  15. A role for glucocorticoid-signaling in depression-like behavior of gastrin-releasing peptide receptor knock-out mice.

    PubMed

    Monje, Francisco J; Kim, Eun-Jung; Cabatic, Maureen; Lubec, Gert; Herkner, Kurt R; Pollak, Daniela D

    2011-08-01

    Abstract Background. The gastrin-releasing peptide receptor (GRPR) is highly expressed in the limbic system, where it importantly regulates emotional functions and in the suprachiasmatic nucleus, where it is central for the photic resetting of the circadian clock. Mice lacking GRPR presented with deficient light-induced phase shift in activity as well altered emotional learning and amygdala function. The effect of GRPR deletion on depression-like behavior and its molecular signature in the amygdala, however, has not yet been evaluated. Methods. GRPR knock-out mice (GRPR-KO) were tested in the forced-swim test and the sucrose preference test for depression-like behavior. Gene expression in the basolateral nucleus of the amygdala was evaluated by micorarray analysis subsequent to laser-capture microdissection-assisted extraction of mRNA. The expression of selected genes was confirmed by RT-PCR. Results. GRPR-KO mice were found to present with increased depression-like behavior. Microarray analysis revealed down-regulation of several glucocorticoid-responsive genes in the basolateral amygdala. Acute administration of dexamethasone reversed the behavioral phenotype and alterations in gene expression. Discussion. We propose that deletion of GRPR leads to the induction of depression-like behavior which is paralleled by dysregulation of amygdala gene expression, potentially resulting from deficient light-induced corticosterone release in GRPR-KO.

  16. Comprehensive Behavioral Phenotyping of Ryanodine Receptor type 3 (RyR3) Knockout Mice: Decreased Social Contact Duration in Two Social Interaction Tests

    PubMed Central

    Matsuo, Naoki; Tanda, Koichi; Nakanishi, Kazuo; Yamasaki, Nobuyuki; Toyama, Keiko; Takao, Keizo; Takeshima, Hiroshi; Miyakawa, Tsuyoshi

    2009-01-01

    Dynamic regulation of the intracellular Ca2+ concentration is crucial for various neuronal functions such as synaptic transmission and plasticity, and gene expression. Ryanodine receptors (RyRs) are a family of intracellular calcium release channels that mediate calcium-induced calcium release from the endoplasmic reticulum. Among the three RyR isoforms, RyR3 is preferentially expressed in the brain especially in the hippocampus and striatum. To investigate the behavioral effects of RyR3 deficiency, we subjected RyR3 knockout (RyR3–/–) mice to a battery of behavioral tests. RyR3–/– mice exhibited significantly decreased social contact duration in two different social interaction tests, where two mice can freely move and make contacts with each other. They also exhibited hyperactivity and mildly impaired prepulse inhibition and latent inhibition while they did not show significant abnormalities in motor function and working and reference memory tests. These results indicate that RyR3 has an important role in locomotor activity and social behavior. PMID:19503748

  17. Androgen Receptor (AR) Physiological Roles in Male and Female Reproductive Systems: Lessons Learned from AR-Knockout Mice Lacking AR in Selective Cells1

    PubMed Central

    Chang, Chawnshang; Lee, Soo Ok; Wang, Ruey-Sheng; Yeh, Shuyuan; Chang, Ta-Min

    2013-01-01

    ABSTRACT Androgens/androgen receptor (AR) signaling is involved primarily in the development of male-specific phenotypes during embryogenesis, spermatogenesis, sexual behavior, and fertility during adult life. However, this signaling has also been shown to play an important role in development of female reproductive organs and their functions, such as ovarian folliculogenesis, embryonic implantation, and uterine and breast development. The establishment of the testicular feminization (Tfm) mouse model exploiting the X-linked Tfm mutation in mice has been a good in vivo tool for studying the human complete androgen insensitivity syndrome, but this mouse may not be the perfect in vivo model. Mouse models with various cell-specific AR knockout (ARKO) might allow us to study AR roles in individual types of cells in these male and female reproductive systems, although discrepancies are found in results between labs, probably due to using various Cre mice and/or knocking out AR in different AR domains. Nevertheless, no doubt exists that the continuous development of these ARKO mouse models and careful studies will provide information useful for understanding AR roles in reproductive systems of humans and may help us to develop more effective and more specific therapeutic approaches for reproductive system-related diseases. PMID:23782840

  18. Nuclear Progestin Receptor (Pgr) Knockouts in Zebrafish Demonstrate Role for Pgr in Ovulation but Not in Rapid Non-Genomic Steroid Mediated Meiosis Resumption

    PubMed Central

    Zhu, Yong; Liu, Dongteng; Shaner, Zoe C.; Chen, Shixi; Hong, Wanshu; Stellwag, Edmund J.

    2015-01-01

    Progestins, progesterone derivatives, are the most critical signaling steroid for initiating final oocyte maturation (FOM) and ovulation, in order to advance fully-grown immature oocytes to become fertilizable eggs in basal vertebrates. It is well-established that progestin induces FOM at least partly through a membrane receptor and a non-genomic steroid signaling process, which precedes progestin triggered ovulation that is mediated through a nuclear progestin receptor (Pgr) and genomic signaling pathway. To determine whether Pgr plays a role in a non-genomic signaling mechanism during FOM, we knocked out Pgr in zebrafish using transcription activator-like effector nucleases (TALENs) and studied the oocyte maturation phenotypes of Pgr knockouts (Pgr-KOs). Three TALENs-induced mutant lines with different frame shift mutations were generated. Homozygous Pgr-KO female fish were all infertile while no fertility effects were evident in homozygous Pgr-KO males. Oocytes developed and underwent FOM normally in vivo in homozygous Pgr-KO female compared to the wild-type controls, but these mature oocytes were trapped within the follicular cells and failed to ovulate from the ovaries. These oocytes also underwent normal germinal vesicle breakdown (GVBD) and FOM in vitro, but failed to ovulate even after treatment with human chronic gonadotropin (HCG) or progestin (17α,20β-dihydroxyprogesterone or DHP), which typically induce FOM and ovulation in wild-type oocytes. The results indicate that anovulation and infertility in homozygous Pgr-KO female fish was, at least in part, due to a lack of functional Pgr-mediated genomic progestin signaling in the follicular cells adjacent to the oocytes. Our study of Pgr-KO supports previous results that demonstrate a role for Pgr in steroid-dependent genomic signaling pathways leading to ovulation, and the first convincing evidence that Pgr is not essential for initiating non-genomic progestin signaling and triggering of meiosis

  19. Characterization of interleukin-1β in Helicobacter pylori-induced gastric inflammation and DNA methylation in interleukin-1 receptor type 1 knockout (IL-1R1(-/-)) mice.

    PubMed

    Huang, Fung-Yu; Chan, Annie On-On; Lo, Regina Cheuk-Lam; Rashid, Asif; Wong, Danny Ka-Ho; Cho, Chi-Hin; Lai, Ching-Lung; Yuen, Man-Fung

    2013-08-01

    Helicobacter pylori infection induced interleukin-1β (IL-1β) production and is associated with aberrant DNA methylation and gastric diseases. Here, we investigated the role of IL-1β in H. pylori-induced gastric inflammation and DNA methylation using IL-1 receptor type 1 knockout (IL-1R1(-/-)) mice, and compared the therapeutic efficacy of antimicrobial therapy with IL-1 receptor antagonist (IL-1ra). IL-1R1(-/-) and wild-type (WT) mice were infected with H. pylori for 16, 24 and 32 weeks. Infected WT mice at 24 weeks were given either antimicrobial therapy or IL-1ra. Comparing to the IL-1R1(-/-) mice, infected WT mice with functional IL-1β signaling had higher gastritis scores, higher IL-1β and iNOS mRNA expression, higher nitric oxide (NO) production and increased frequency of E-cadherin (E-cad) methylation at all the time points analyzed. IL-1β release was significantly elevated in infected WT mice than normal controls at 16 weeks post-infection (p<0.005). Treatment of infected mice with antimicrobial therapy and IL-1ra significantly reduced the degree of gastritis (p<0.005; p<0.05, respectively), iNOS expression (p<0.0001; p<0.01, respectively) and NO production (both p<0.001) compared with untreated controls. Mice receiving antimicrobial therapy had significantly lower IL-1β expression than untreated controls (p<0.0001). Both treatments reduced the incidence of E-cad methylation in infected mice compared with controls, however, no statistical significance was observed. There was no significant alteration of total DNA methyltransferase (DNMT) activity. These results demonstrated that IL-1β played a crucial role in H. pylori-induced gastric inflammation and DNA methylation. H. pylori eradication and IL-1ra administration could ameliorate inflammatory stress.

  20. Automated pipeline to analyze non-contact infrared images of the paraventricular nucleus specific leptin receptor knock-out mouse model

    NASA Astrophysics Data System (ADS)

    Diaz Martinez, Myriam; Ghamari-Langroudi, Masoud; Gifford, Aliya; Cone, Roger; Welch, E. B.

    2015-03-01

    Evidence of leptin resistance is indicated by elevated leptin levels together with other hallmarks of obesity such as a defect in energy homeostasis.1 As obesity is an increasing epidemic in the US, the investigation of mechanisms by which leptin resistance has a pathophysiological impact on energy is an intensive field of research.2 However, the manner in which leptin resistance contributes to the dysregulation of energy, specifically thermoregulation,3 is not known. The aim of this study was to investigate whether the leptin receptor expressed in paraventricular nucleus (PVN) neurons plays a role in thermoregulation at different temperatures. Non-contact infrared (NCIR) thermometry was employed to measure surface body temperature (SBT) of nonanesthetized mice with a specific deletion of the leptin receptor in the PVN after exposure to room (25 °C) and cold (4 °C) temperature. Dorsal side infrared images of wild type (LepRwtwt/sim1-Cre), heterozygous (LepRfloxwt/sim1-Cre) and knock-out (LepRfloxflox/sim1-Cre) mice were collected. Images were input to an automated post-processing pipeline developed in MATLAB to calculate average and maximum SBTs. Linear regression was used to evaluate the relationship between sex, cold exposure and leptin genotype with SBT measurements. Findings indicate that average SBT has a negative relationship to the LepRfloxflox/sim1-Cre genotype, the female sex and cold exposure. However, max SBT is affected by the LepRfloxflox/sim1-Cre genotype and the female sex. In conclusion this data suggests that leptin within the PVN may have a neuroendocrine role in thermoregulation and that NCIR thermometry combined with an automated imaging-processing pipeline is a promising approach to determine SBT in non-anesthetized mice.

  1. Adult female wildtype, but not oestrogen receptor β knockout, mice have decreased depression-like behaviour during pro-oestrus and following administration of oestradiol or diarylpropionitrile

    PubMed Central

    Walf, AA; Koonce, CJ; Frye, CA

    2013-01-01

    Studies in people and animal models suggest that depression is influenced by natural, fluctuations in the levels of 17β-oestradiol (E2), as well as administration of E2-based therapies, such as selective oestrogen receptor modulators (SERMs). Elucidating the effects and mechanisms of E2 is important to improve future E2-based therapeutics. An important question is whether effects of E2 or SERMs for mood regulation act at the α or β isoform of the oestrogen receptor (ER) because some of the unwanted trophic effects of E2-based therapies may involve actions at ERα, rather than ERβ. In the present study, whether there are sex differences in depression-like behaviour of adult mice (experiment 1), and the effects of natural fluctuations in E2 (experiment 2), or administration of E2 or a SERM that has higher affinity for ERβ than for ERα (diarylpropionitrile; DPN) to ovariectomised (experiment 3) wildtype and ERβ knockout (βERKO) mice were investigated. Results of this study supported our hypotheses that: there would be sex differences favouring males for depression-like behaviour and endogenous increases in, or exogenous administration of, E2 or administration of an ERβ SERM would decrease depression-like behaviour in wildtype, but not βERKO, mice. In experiment 1, adult male mice spent less time immobile in the forced swim test (i.e., showed less depression-like behaviour) compared with female mice. In experiment 2, pro-oestrous (higher circulating E2 levels), compared with dioestrous (lower circulating E2 levels), mice had reduced immobility in the forced swim test; this effect was not observed in βERKO mice. In experiment 3, administration of E2 or DPN to ovariectomised wildtype, but not βERKO, mice decreased immobility compared with vehicle administration, these data suggest that ERβ may be required for some of the anti–depressant-like effects of E2. PMID:18562442

  2. Adipose tissue deficiency results in severe hyperlipidemia and atherosclerosis in the low-density lipoprotein receptor knockout mice.

    PubMed

    Wang, Mengyu; Gao, Mingming; Liao, Jiawei; Qi, Yanfei; Du, Ximing; Wang, Yuhui; Li, Ling; Liu, George; Yang, Hongyuan

    2016-05-01

    Adipose tissue can store over 50% of whole-body cholesterol; however, the physiological role of adipose tissue in cholesterol metabolism and atherogenesis has not been directly assessed. Here, we examined lipoprotein metabolism and atherogenesis in a unique mouse model of severe lipodystrophy: the Seipin(-/-) mice, and also in mice deficient in both low-density lipoprotein receptor (Ldlr) and Seipin: the Ldlr(-/-)Seipin(-/-) mice. Plasma cholesterol was moderately increased in the Seipin(-/-) mice when fed an atherogenic diet. Strikingly, plasma cholesterol reached ~6000 mg/dl in the Seipin(-/-)Ldlr(-/-) mice on an atherogenic diet, as compared to ~1000 mg/dl in the Ldlr(-/-) mice on the same diet. The Seipin(-/-)Ldlr(-/-) mice also developed spontaneous atherosclerosis on chow diet and severe atherosclerosis on an atherogenic diet. Rosiglitazone treatment significantly reduced the hypercholesterolemia of the Seipin(-/-)Ldlr(-/-) mice, and also alleviated the severity of atherosclerosis. Our results provide direct evidence, for the first time, that the adipose tissue plays a critical role in the clearance of plasma cholesterol. Our results also reveal a previously unappreciated strong link between adipose tissue and LDLR in plasma cholesterol metabolism.

  3. Discovery of 2-substituted benzoxazole carboxamides as 5-HT3 receptor antagonists.

    PubMed

    Yang, Zhicai; Fairfax, David J; Maeng, Jun-Ho; Masih, Liaqat; Usyatinsky, Alexander; Hassler, Carla; Isaacson, Soshanna; Fitzpatrick, Kevin; DeOrazio, Russell J; Chen, Jianqing; Harding, James P; Isherwood, Matthew; Dobritsa, Svetlana; Christensen, Kevin L; Wierschke, Jonathan D; Bliss, Brian I; Peterson, Lisa H; Beer, Cathy M; Cioffi, Christopher; Lynch, Michael; Rennells, W Martin; Richards, Justin J; Rust, Timothy; Khmelnitsky, Yuri L; Cohen, Marlene L; Manning, David D

    2010-11-15

    A new class of 2-substituted benzoxazole carboxamides are presented as potent functional 5-HT(3) receptor antagonists. The chemical series possesses nanomolar in vitro activity against human 5-HT(3)A receptors. A chemistry optimization program was conducted and identified 2-aminobenzoxazoles as orally active 5-HT(3) receptor antagonists with good metabolic stability. These novel analogues possess drug-like characteristics and have potential utility for the treatment of diseases attributable to improper 5-HT(3) receptor function, especially diarrhea predominant irritable bowel syndrome (IBS-D).

  4. Nociceptin/orphanin FQ (N/OFQ)-evoked bradycardia, hypotension, and diuresis are absent in N/OFQ peptide (NOP) receptor knockout mice.

    PubMed

    Burmeister, Melissa A; Ansonoff, Michael A; Pintar, John E; Kapusta, Daniel R

    2008-09-01

    Intracerebroventricular administration of the opioid-like peptide nociceptin/orphanin FQ (N/OFQ) produces bradycardia, hypotension, and diuresis in mice. We hypothesized that these responses are solely caused by selective activation of central N/OFQ peptide (NOP) receptors. To test this premise, we first examined whether i.c.v. N/OFQ produced dose-dependent diuretic and cardiovascular depressor responses in commercially available C57BL/6 mice. Next, using doses established in these studies, we examined the renal excretory and cardiovascular responses to i.c.v. N/OFQ in conscious transgenic NOP receptor knockout mice (NOP(-/-)). In metabolic studies, i.c.v. N/OFQ, but not saline vehicle, dose-dependently increased urine output (V) in NOP(+/+); this response was significant at 3 nmol (N/OFQ, V = 0.39 +/- 0.10 ml/2 h; saline, 0.08 +/- 0.05 ml/2 h). The N/OFQ-evoked diuresis was absent in littermate NOP(-/-) (N/OFQ, V = 0.06 +/- 0.06 ml/2 h; saline, 0.03 +/- 0.03 ml/2 h). There were no significant changes in urinary sodium or potassium excretion or free water clearance in either group. In telemetry studies, i.c.v. N/OFQ dose dependently lowered heart rate (HR) and mean arterial pressure (MAP). At 3 nmol N/OFQ, both HR and MAP were reduced in NOP(+/+) (peak DeltaHR = -217 +/- 31 bpm; peak DeltaMAP =-47 +/- 7 mm Hg) compared with saline (peak DeltaHR =-14 +/- 5 bpm; peak DeltaMAP = 2 +/- 3 mm Hg). These N/OFQ-evoked bradycardic and hypotensive responses were absent in NOP(-/-) (peak DeltaHR =-13 +/- 17 bpm; peak DeltaMAP =-2 +/- 4 mm Hg, respectively). Basal 24-h cardiovascular and renal excretory function were not different between NOP(-/-) and NOP(+/+) mice. These results establish that the bradycardia, hypotension and diuresis produced by centrally administered N/OFQ are mediated by selective activation of NOP receptors.

  5. Epididymal Hypo-Osmolality Induces Abnormal Sperm Morphology and Function in the Estrogen Receptor Alpha Knockout Mouse1

    PubMed Central

    Joseph, Avenel; Shur, Barry D.; Ko, CheMyong; Chambon, Pierre; Hess, Rex A.

    2010-01-01

    Estrogen receptor-alpha (ESR1) is highly expressed in the efferent ductules of all species studied as well as in the epididymal epithelium in mice and other select species. Male mice lacking ESR1 (Esr1KO) are infertile, but transplantation studies demonstrated that Esr1KO germ cells are capable of fertilization when placed in a wild-type reproductive tract. These results suggest that extratesticular regions, such as the efferent ductules and epididymis, are the major source of pathological changes in Esr1KO males. Previous studies have shown alterations in ion and fluid transporters in the efferent duct and epididymal epithelia of Esr1KO males, leading to misregulation of luminal fluid pH. To determine the effect of an altered epididymal milieu on Esr1KO sperm, we assayed sperm morphology in the different regions of the epididymis. Sperm recovered from the epididymis exhibited abnormal flagellar coiling and increased incidence of spontaneous acrosome reactions, both of which are consistent with exposure to abnormal epididymal fluid. Analysis of the epididymal fluid revealed that the osmolality of the Esr1KO fluid was reduced relative to wild type, consistent with prior reports of inappropriate fluid absorption from the efferent ductules. This, along with the finding that morphological defects increased with transit through the epididymal duct, suggests that the anomalies in sperm are a consequence of the abnormal luminal environment. Consistent with this, incubating Esr1KO sperm in a more wild-type-like osmotic environment significantly rescued the abnormal flagellar coiling. This work demonstrates that Esr1KO mice exhibit an abnormal fluid environment in the lumen of the efferent ducts and epididymis, precluding normal sperm maturation and instead resulting in progressive deterioration of sperm that contributes to infertility. PMID:20130266

  6. Increased oxidative stress and apoptosis in the hypothalamus of diabetic male mice in the insulin receptor substrate-2 knockout model

    PubMed Central

    Canelles, Sandra; Argente, Jesús; Barrios, Vicente

    2016-01-01

    ABSTRACT Insulin receptor substrate-2-deficient (IRS2−/−) mice are considered a good model to study the development of diabetes because IRS proteins mediate the pleiotropic effects of insulin-like growth factor-I (IGF-I) and insulin on metabolism, mitogenesis and cell survival. The hypothalamus might play a key role in the early onset of diabetes, owing to its involvement in the control of glucose homeostasis and energy balance. Because some inflammatory markers are elevated in the hypothalamus of diabetic IRS2−/− mice, our aim was to analyze whether the diabetes associated with the absence of IRS2 results in hypothalamic injury and to analyze the intracellular mechanisms involved. Only diabetic IRS2−/− mice showed increased cell death and activation of caspase-8 and -3 in the hypothalamus. Regulators of apoptosis such as FADD, Bcl-2, Bcl-xL and p53 were also increased, whereas p-IκB and c-FLIPL were decreased. This was accompanied by increased levels of Nox-4 and catalase, enzymes involved in oxidative stress. In summary, the hypothalamus of diabetic IRS2−/− mice showed an increase in oxidative stress and inflammatory markers that finally resulted in cell death via substantial activation of the extrinsic apoptotic pathway. Conversely, non-diabetic IRS2−/− mice did not show cell death in the hypothalamus, possibly owing to an increase in the levels of circulating IGF-I and in the enhanced hypothalamic IGF-IR phosphorylation that would lead to the stimulation of survival pathways. In conclusion, diabetes in IRS2-deficient male mice is associated with increased oxidative stress and apoptosis in the hypothalamus. PMID:27013528

  7. Increased oxidative stress and apoptosis in the hypothalamus of diabetic male mice in the insulin receptor substrate-2 knockout model.

    PubMed

    Baquedano, Eva; Burgos-Ramos, Emma; Canelles, Sandra; González-Rodríguez, Agueda; Chowen, Julie A; Argente, Jesús; Barrios, Vicente; Valverde, Angela M; Frago, Laura M

    2016-05-01

    Insulin receptor substrate-2-deficient (IRS2(-/-)) mice are considered a good model to study the development of diabetes because IRS proteins mediate the pleiotropic effects of insulin-like growth factor-I (IGF-I) and insulin on metabolism, mitogenesis and cell survival. The hypothalamus might play a key role in the early onset of diabetes, owing to its involvement in the control of glucose homeostasis and energy balance. Because some inflammatory markers are elevated in the hypothalamus of diabetic IRS2(-/-) mice, our aim was to analyze whether the diabetes associated with the absence of IRS2 results in hypothalamic injury and to analyze the intracellular mechanisms involved. Only diabetic IRS2(-/-) mice showed increased cell death and activation of caspase-8 and -3 in the hypothalamus. Regulators of apoptosis such as FADD, Bcl-2, Bcl-xL and p53 were also increased, whereas p-IκB and c-FLIPL were decreased. This was accompanied by increased levels of Nox-4 and catalase, enzymes involved in oxidative stress. In summary, the hypothalamus of diabetic IRS2(-/-) mice showed an increase in oxidative stress and inflammatory markers that finally resulted in cell death via substantial activation of the extrinsic apoptotic pathway. Conversely, non-diabetic IRS2(-/-) mice did not show cell death in the hypothalamus, possibly owing to an increase in the levels of circulating IGF-I and in the enhanced hypothalamic IGF-IR phosphorylation that would lead to the stimulation of survival pathways. In conclusion, diabetes in IRS2-deficient male mice is associated with increased oxidative stress and apoptosis in the hypothalamus. PMID:27013528

  8. Increased oxidative stress and apoptosis in the hypothalamus of diabetic male mice in the insulin receptor substrate-2 knockout model.

    PubMed

    Baquedano, Eva; Burgos-Ramos, Emma; Canelles, Sandra; González-Rodríguez, Agueda; Chowen, Julie A; Argente, Jesús; Barrios, Vicente; Valverde, Angela M; Frago, Laura M

    2016-05-01

    Insulin receptor substrate-2-deficient (IRS2(-/-)) mice are considered a good model to study the development of diabetes because IRS proteins mediate the pleiotropic effects of insulin-like growth factor-I (IGF-I) and insulin on metabolism, mitogenesis and cell survival. The hypothalamus might play a key role in the early onset of diabetes, owing to its involvement in the control of glucose homeostasis and energy balance. Because some inflammatory markers are elevated in the hypothalamus of diabetic IRS2(-/-) mice, our aim was to analyze whether the diabetes associated with the absence of IRS2 results in hypothalamic injury and to analyze the intracellular mechanisms involved. Only diabetic IRS2(-/-) mice showed increased cell death and activation of caspase-8 and -3 in the hypothalamus. Regulators of apoptosis such as FADD, Bcl-2, Bcl-xL and p53 were also increased, whereas p-IκB and c-FLIPL were decreased. This was accompanied by increased levels of Nox-4 and catalase, enzymes involved in oxidative stress. In summary, the hypothalamus of diabetic IRS2(-/-) mice showed an increase in oxidative stress and inflammatory markers that finally resulted in cell death via substantial activation of the extrinsic apoptotic pathway. Conversely, non-diabetic IRS2(-/-) mice did not show cell death in the hypothalamus, possibly owing to an increase in the levels of circulating IGF-I and in the enhanced hypothalamic IGF-IR phosphorylation that would lead to the stimulation of survival pathways. In conclusion, diabetes in IRS2-deficient male mice is associated with increased oxidative stress and apoptosis in the hypothalamus.

  9. Conditional knockout of collecting duct bradykinin B2 receptors exacerbates angiotensin II-induced hypertension during high salt intake.

    PubMed

    Kopkan, Libor; Husková, Zuzana; Jíchová, Šárka; Červenková, Lenka; Červenka, Luděk; Saifudeen, Zubaida; El-Dahr, Samir S

    2016-01-01

    We elucidated the role of collecting duct kinin B2 receptor (B2R) in the development of salt-sensitivity and angiotensin II (ANG II)-induced hypertension. To this end, we used a Cre-Lox recombination strategy to generate mice lacking Bdkrb2 gene for B2R in the collecting duct (Hoxb7-Cre(tg/+):Bdkrb2(flox/flox)). In 3 groups of control (Bdkrb2(flox/flox)) and 3 groups of UB(Bdkrb2-/-) mice, systolic blood pressure (SBP) responses to high salt intake (4 or 8% NaCl; HS) were monitored by radiotelemetry in comparison with standard salt diet (0.4% NaCl) prior to and during subcutaneous ANG II infusion (1000 ng/min/kg) via osmotic minipumps. High salt intakes alone for 2 weeks did not alter SBP in either strain. ANG II significantly increased SBP equally in control (121 ± 2 to 156 ± 3 mmHg) and UB(Bdkrb2-/-) mice (120 ± 2 to 153 ± 2 mmHg). The development of ANG II-induced hypertension was exacerbated by 4%HS in both control (125 ± 3 to 164 ± 5 mmHg) and UB(Bdkrb2-/-) mice (124 ± 2 to 162 ± 3 mmHg) during 2 weeks. Interestingly, 8%HS caused a more profound and earlier ANG II-induced hypertension in UB(Bdkrb2-/-) (129 ± 2 to 166 ± 3 mmHg) as compared to control (128 ± 2 to 158 ± 2 mmHg) and it was accompanied by body weight loss and increased mortality. In conclusion, targeted inactivation of B2R in the renal collecting duct does not cause salt-sensitivity; however, collecting duct B2R attenuates the hypertensive actions of ANG II under conditions of very high salt intake.

  10. Angiotensin receptor-1A knockout leads to hydronephrosis not associated with a loss of pyeloureteric peristalsis in the mouse renal pelvis.

    PubMed

    Nguyen, Michael J; Hashitani, Hikaru; Lang, Richard J

    2016-05-01

    The action of angiotensin II (AngII) on the Ca(2+) signals driving pyeloureteric peristalsis was investigated using both conventional and angiotensin receptor (ATr) ATr1A and ATr2 knockout ((-/-)) mice. Contractility in the renal pelvis of adult ATr1A(-/-) and ATr2(-/-) mice was compared to their respective wildtype (ATr1A(+/+) and ATr2(+/+)) controls of the same genetic background (FVB/N and C57Bl/6 respectively) using video microscopy. The effects of AngII on the Ca(2+) signals in typical and atypical smooth muscle cells (TSMCs and ASMCs, respectively) within the pelvic wall of conventional mice were recorded using Fluo-4 Ca(2+) imaging. Compared to ATr1A(+/+) , ATr2(+/+) and ATr2(-/-) mice, kidneys of the ATr1A(-/-) mouse were mildly-to-severely hydronephrotic, associated with an enlarged calyx, an atrophic papilla and a hypoplastic renal pelvis. Contraction frequencies in the renal pelvis of moderately hydronephrotic ATr1A(-/-) and unaffected ATr2(-/-) mice were not significantly different from their ATr1A(+/+), ATr2(+/+) controls. No contractions were observed in severely-hydronephrotic ATr1A(-/-) kidneys. AngII increased the spontaneous contraction frequency of the renal pelvis in ATr1A(+/+), ATr2(+/+) and ATr2(-/-) mice, but had little effect on the contractions in the mildly-hydronephrotic ATr1A(-/-) renal pelvis. The ATr1 blocker, candesartan prevented the positive chronotropic effects of AngII. AngII increased the frequency and synchronicity of Ca(2+) transients in both TSMCs and ASMCs. It was concluded that the hydronephrosis observed in ATr1A(-/-) mouse kidneys does not arise from a failure in the development of the essential pacemaker and contractile machinery driving pyeloureteric peristalsis. PMID:26876143

  11. High Fat High Cholesterol Diet (Western Diet) Aggravates Atherosclerosis, Hyperglycemia and Renal Failure in Nephrectomized LDL Receptor Knockout Mice: Role of Intestine Derived Lipopolysaccharide.

    PubMed

    Ghosh, Siddhartha S; Righi, Samuel; Krieg, Richard; Kang, Le; Carl, Daniel; Wang, Jing; Massey, H Davis; Sica, Domenic A; Gehr, Todd W B; Ghosh, Shobha

    2015-01-01

    A high fat meal, frequently known as western diet (WD), exacerbates atherosclerosis and diabetes. Both these diseases are frequently associated with renal failure. Recent studies have shown that lipopolysaccharide (LPS) leaks into the circulation from the intestine in the setting of renal failure and after WD. However, it is not clear how renal function and associated disorders are affected by LPS. This study demonstrates that circulatory LPS exacerbates renal insufficiency, atherosclerosis and glucose intolerance. Renal insufficiency was induced by 2/3 nephrectomy in LDL receptor knockout mice. Nx animals were given normal diet (Nx) or WD (Nx+WD). The controls were sham operated animals on normal diet (control) and WD (WD). To verify if LPS plays a role in exaggerating renal insufficiency, polymyxin (PM), a known LPS antagonist, and curcumin (CU), a compound known to ameliorate chronic kidney disease (CKD), was given to Nx animals on western diet (Nx+WD+PM and Nx+WD+CU, respectively). Compared to control, all other groups displayed increased circulatory LPS. The Nx+WD cohort had the highest levels of LPS. Nx group had significant renal insufficiency and glucose intolerance but not atherosclerosis. WD had intense atherosclerosis and glucose intolerance but it did not show signs of renal insufficiency. Compared to other groups, Nx+WD had significantly higher cytokine expression, macrophage infiltration in the kidney, renal insufficiency, glucose intolerance and atherosclerosis. PM treatment blunted the expression of cytokines, deterioration of renal function and associated disorders, albeit not to the levels of Nx, and was significantly inferior to CU. PM is a non-absorbable antibiotic with LPS binding properties, hence its beneficial effect can only be due to its effect within the GI tract. We conclude that LPS may not cause renal insufficiency but can exaggerate kidney failure and associated disorders following renal insufficiency.

  12. Experimental transmission of AA amyloidosis by injecting the AA amyloid protein into interleukin-1 receptor antagonist knockout (IL-1raKO) mice.

    PubMed

    Watanabe, K; Uchida, K; Chambers, J K; Tei, M; Shoji, A; Ushio, N; Nakayama, H

    2015-05-01

    The incidence of AA amyloidosis is high in humans with rheumatoid arthritis and several animal species, including cats and cattle with prolonged inflammation. AA amyloidosis can be experimentally induced in mice using severe inflammatory stimuli and a coinjection of AA amyloid; however, difficulties have been associated with transmitting AA amyloidosis to a different animal species, and this has been attributed to the "species barrier." The interleukin-1 receptor antagonist knockout (IL-1raKO) mouse, a rodent model of human rheumatoid arthritis, has been used in the transmission of AA amyloid. When IL-1raKO and BALB/c mice were intraperitoneally injected with mouse AA amyloid together with a subcutaneous pretreatment of 2% AgNO3, all mice from both strains that were injected with crude or purified murine AA amyloid developed AA amyloidosis. However, the amyloid index, which was determined by the intensity of AA amyloid deposition, was significantly higher in IL-1raKO mice than in BALB/c mice. When IL-1raKO and BALB/c mice were injected with crude or purified bovine AA amyloid together with the pretreatment, 83% (5/6 cases) and 38% (3/8 cases) of IL-1raKO mice and 17% (1/6 cases) and 0% (0/6 cases) of BALB/c mice, respectively, developed AA amyloidosis. Similarly, when IL-1raKO and BALB/c mice were injected with crude or purified feline AA amyloid, 33% (2/6 cases) and 88% (7/8 cases) of IL-1raKO mice and 0% (0/6 cases) and 29% (2/6 cases) of BALB/c mice, respectively, developed AA amyloidosis. These results indicated that IL-1raKO mice are a useful animal model for investigating AA amyloidogenesis.

  13. High Fat High Cholesterol Diet (Western Diet) Aggravates Atherosclerosis, Hyperglycemia and Renal Failure in Nephrectomized LDL Receptor Knockout Mice: Role of Intestine Derived Lipopolysaccharide

    PubMed Central

    Ghosh, Siddhartha S.; Righi, Samuel; Krieg, Richard; Kang, Le; Carl, Daniel; Wang, Jing; Massey, H. Davis; Sica, Domenic A.; Gehr, Todd W. B.; Ghosh, Shobha

    2015-01-01

    A high fat meal, frequently known as western diet (WD), exacerbates atherosclerosis and diabetes. Both these diseases are frequently associated with renal failure. Recent studies have shown that lipopolysaccharide (LPS) leaks into the circulation from the intestine in the setting of renal failure and after WD. However, it is not clear how renal function and associated disorders are affected by LPS. This study demonstrates that circulatory LPS exacerbates renal insufficiency, atherosclerosis and glucose intolerance. Renal insufficiency was induced by 2/3 nephrectomy in LDL receptor knockout mice. Nx animals were given normal diet (Nx) or WD (Nx+WD). The controls were sham operated animals on normal diet (control) and WD (WD). To verify if LPS plays a role in exaggerating renal insufficiency, polymyxin (PM), a known LPS antagonist, and curcumin (CU), a compound known to ameliorate chronic kidney disease (CKD), was given to Nx animals on western diet (Nx+WD+PM and Nx+WD+CU, respectively). Compared to control, all other groups displayed increased circulatory LPS. The Nx+WD cohort had the highest levels of LPS. Nx group had significant renal insufficiency and glucose intolerance but not atherosclerosis. WD had intense atherosclerosis and glucose intolerance but it did not show signs of renal insufficiency. Compared to other groups, Nx+WD had significantly higher cytokine expression, macrophage infiltration in the kidney, renal insufficiency, glucose intolerance and atherosclerosis. PM treatment blunted the expression of cytokines, deterioration of renal function and associated disorders, albeit not to the levels of Nx, and was significantly inferior to CU. PM is a non-absorbable antibiotic with LPS binding properties, hence its beneficial effect can only be due to its effect within the GI tract. We conclude that LPS may not cause renal insufficiency but can exaggerate kidney failure and associated disorders following renal insufficiency. PMID:26580567

  14. Pentameric quaternary structure of the intracellular domain of serotonin type 3A receptors

    PubMed Central

    Pandhare, Akash; Grozdanov, Petar N.; Jansen, Michaela

    2016-01-01

    In spite of extensive efforts over decades an experimentally-derived structure of full-length eukaryotic pentameric ligand-gated ion channels (pLGICs) is still lacking. These pharmaceutically highly-relevant channels contain structurally well-conserved and characterized extracellular and transmembrane domains. The intracellular domain (ICD), however, has been orphaned in structural studies based on the consensus assumption of being largely disordered. In the present study, we demonstrate for the first time that the serotonin type 3A (5-HT3A) ICD assembles into stable pentamers in solution in the absence of the other two domains, thought to be the drivers for oligomerization. Additionally, the soluble 5-HT3A-ICD construct interacted with the protein RIC-3 (resistance to inhibitors of cholinesterase). The interaction provides evidence that the 5-HT3A-ICD is not only required but also sufficient for interaction with RIC-3. Our results suggest the ICD constitutes an oligomerization domain. This novel role significantly adds to its known contributions in receptor trafficking, targeting, and functional fine-tuning. The innate diversity of the ICDs with sizes ranging from 50 to 280 amino acids indicates new methodologies need to be developed to determine the structures of these domains. The use of soluble ICD proteins that we report in the present study constitutes a useful approach to address this gap. PMID:27045630

  15. Normal thermoregulatory responses to 3-iodothyronamine, trace amines and amphetamine-like psychostimulants in trace amine associated receptor 1 knockout mice.

    PubMed

    Panas, Helen N; Lynch, Laurie J; Vallender, Eric J; Xie, Zhihua; Chen, Guo-Lin; Lynn, Spencer K; Scanlan, Thomas S; Miller, Gregory M

    2010-07-01

    3-Iodothyronamine (T1AM) is a metabolite of thyroid hormone. It is an agonist at trace amine-associated receptor 1 (TAAR1), a recently identified receptor involved in monoaminergic regulation and a potential novel therapeutic target. Here, T1AM was studied using rhesus monkey TAAR1 and/or human dopamine transporter (DAT) co-transfected cells, and wild-type (WT) and TAAR1 knock-out (KO) mice. The IC(50) of T1AM competition for binding of the DAT-specific radio-ligand [(3)H]CFT was highly similar in DAT cells, WT striatal synaptosomes and KO striatal synaptosomes (0.72-0.81 microM). T1AM inhibition of 10 nM [(3)H]dopamine uptake (IC(50): WT, 1.4 + or - 0.5 microM; KO, 1.2 + or - 0.4 microM) or 50 nM [(3)H]serotonin uptake (IC(50): WT, 4.5 + or - 0.6 microM; KO, 4.7 + or - 1.1 microM) in WT and KO synaptosomes was also highly similar. Unlike other TAAR1 agonists that are DAT substrates, TAAR1 signaling in response to T1AM was not enhanced in the presence of DAT as determined by CRE-luciferase assay. In vivo, T1AM induced robust hypothermia in WT and KO mice equivalently and dose dependently (maximum change degrees Celsius: 50 mg/kg at 60 min: WT -6.0 + or - 0.4, KO -5.6 + or - 1.0; and 25 mg/kg at 30 min: WT -2.7 + or - 0.4, KO -3.0 + or - 0.2). Other TAAR1 agonists including beta-phenylethylamine (beta-PEA), MDMA (3,4-methylenedioxymethamphetamine) and methamphetamine also induced significant, time-dependent thermoregulatory responses that were alike in WT and KO mice. Therefore, TAAR1 co-expression does not alter T1AM binding to DAT in vitro nor T1AM inhibition of [(3)H]monoamine uptake ex vivo, and TAAR1 agonist-induced thermoregulatory responses are TAAR1-independent. Accordingly, TAAR1-directed compounds will likely not affect thermoregulation nor are they likely to be cryogens.

  16. Role of α7- and β4-containing nicotinic acetylcholine receptors in the affective and somatic aspects of nicotine withdrawal: studies in knockout mice.

    PubMed

    Stoker, Astrid K; Olivier, Berend; Markou, Athina

    2012-05-01

    To assess which nicotinic acetylcholine receptors (nAChRs) are involved in the aversive aspects of nicotine withdrawal, brain reward function and the somatic signs of nicotine withdrawal were assessed in mice that lack α7 and β4 nAChR subunits. Brain reward function was assessed with the intracranial self-stimulation (ICSS) procedure, in which elevations in ICSS thresholds reflect an anhedonic mood state. At 3-6 h of spontaneous nicotine/saline withdrawal, thresholds were elevated in nicotine-withdrawing α7(+/+) and β4(+/+), but not α7(-/-) or β4(-/-), mice compared with saline-withdrawing mice, indicating a delay in the onset of withdrawal in the knockout mice. From 8 to 100 h of withdrawal, thresholds in α7(+/+) and α7(-/-) mice were equally elevated, whereas thresholds in β4(+/+) and β4(-/-) mice returned to baseline levels. Somatic signs were attenuated in nicotine-withdrawing β4(-/-), but not α7(-/-), mice. Administration of a low dose of the nAChR antagonist mecamylamine induced threshold elevations in α7(-/-), but not α7(+/+), mice, whereas the highest dose tested only elevated thresholds in α7(+/+) mice. Mecamylamine-induced threshold elevations were similar in β4(-/-) and β4(+/+) mice. In conclusion, null mutation of the α7 and β4 nAChR subunits resulted in a delayed onset of the anhedonic aspects of the spontaneous nicotine withdrawal syndrome. Previous findings of attenuated somatic signs of nicotine withdrawal in β4(-/-), but not α7(-/-), mice were confirmed in the present study, indicating an important role for β4-containing nAChRs in the somatic signs of nicotine withdrawal. The mecamylamine-precipitated withdrawal data suggest that compensatory adaptations may occur in constitutive α7(-/-) mice or that mecamylamine may interact with other receptors besides nAChRs in these mice. In summary, the present results indicate an important role for α7 and β4-containing nAChRs in the anhedonic or somatic signs of nicotine withdrawal.

  17. GH in the dwarf dopaminergic D2 receptor knockout mouse: somatotrope population, GH release, and responsiveness to GH-releasing factors and somatostatin.

    PubMed

    García-Tornadú, Isabel; Rubinstein, Marcelo; Gaylinn, Bruce D; Hill, David; Arany, Edith; Low, Malcolm J; Díaz-Torga, Graciela; Becu-Villalobos, Damasia

    2006-09-01

    Recently, the importance of the dopaminergic D2 receptor (D2R) subtype in normal body growth and neonatal GH secretion has been highlighted. Disruption of D2R alters the GHRH-GH-IGF-I axis and impairs body growth in adult male mice. The D2R knockout (KO) dwarf mouse has not been well characterized; we therefore sought to determine somatotrope function in the adult pituitary. Using immunohistochemistry and confocal microscopy, we found a significant decrease in the somatotrope population in pituitaries from KO mice (P=0.043), which was paralleled by a decreased GH output from pituitary cells cultured in vitro. In cells from adult mice the response amplitude to GHRH differed between genotypes (lower in KO), but this difference was less dramatic after taking into account the lower basal release and hormone content in the KO cells. Furthermore, there were no significant differences in cAMP generation in response to GHRH between genotypes. By Western blot, GHRH-receptor in pituitary membranes from KO mice was reduced to 46% of the level found in wildtype (WT) mice (P=0.016). Somatostatin induced a concentration-dependent decrease in GH and prolactin (PRL) secretion in both genotypes, and 1x10(-7) M ghrelin released GH in cells from both genotypes (P=0.017) in a proportionate manner to basal levels. These results suggest that KO somatotropes maintain a regulated secretory function. Finally, we tested the direct effect of dopamine on GH and PRL secretion in cells from both genotypes at 20 days and 6 months of life. As expected, we found that dopamine could reduce PRL levels at both ages in WT mice but not in KO mice, but there was no consistent effect of the neurotransmitter on GH release in either genotype at the ages studied. The present study demonstrates that in the adult male D2R KO mouse, there is a reduction in pituitary GH content and secretory activity. Our results point to an involvement of D2R signaling at the hypothalamic level as dopamine did not release GH

  18. Physiological roles of A1 and A2A adenosine receptors in regulating heart rate, body temperature, and locomotion as revealed using knockout mice and caffeine.

    PubMed

    Yang, Jiang-Ning; Chen, Jiang-Fan; Fredholm, Bertil B

    2009-04-01

    Heart rate (HR), body temperature (Temp), locomotor activity (LA), and oxygen consumption (O(2)C) were studied in awake mice lacking one or both of the adenosine A(1) or A(2A) receptors (A(1)R or A(2A)R, respectively) using telemetry and respirometry, before and after caffeine administration. All parameters were lower during day than night and higher in females than males. When compared with wild-type (WT) littermates, HR was higher in male A(1)R knockout (A(1)RKO) mice but lower in A(2A)RKO mice and intermediate in A(1)-A(2A)R double KO mice. A single dose of an unselective beta-blocker (timolol; 1 mg/kg) abolished the HR differences between these genotypes. Deletion of A(1)Rs had little effect on Temp, whereas deletion of A(2A)Rs increased it in females and decreased it in males. A(1)-A(2A)RKO mice had lower Temp than WT mice. LA was unaltered in A(1)RKO mice and lower in A(2A)RKO and A(1)-A(2A)RKO mice than in WT mice. Caffeine injection increased LA but only in mice expressing A(2A)R. Caffeine ingestion also increased LA in an A(2A)R-dependent manner in male mice. Caffeine ingestion significantly increased O(2)C in WT mice, but less in the different KO mice. Injection of 30 mg/kg caffeine decreased Temp, especially in KO mice, and hence in a manner unrelated to A(1)R or A(2A)R blockade. Selective A(2B) antagonism had little or no effect. Thus A(1)R and A(2A)R influence HR, Temp, LA, and O(2)C in mice in a sex-dependent manner, indicating effects of endogenous adenosine. The A(2A)R plays an important role in the modulation of O(2)C and LA by acute and chronic caffeine administration. There is also evidence for effects of higher doses of caffeine being independent of both A(1)R and A(2A)R.

  19. Oleylphosphocholine (OlPC) arrests Cryptosporidium parvum growth in vitro and prevents lethal infection in interferon gamma receptor knock-out mice

    PubMed Central

    Sonzogni-Desautels, Karine; Renteria, Axel E.; Camargo, Fabio V.; Di Lenardo, Thomas Z.; Mikhail, Alexandre; Arrowood, Michael J.; Fortin, Anny; Ndao, Momar

    2015-01-01

    Cryptosporidium parvum is a species of protozoa that causes cryptosporidiosis, an intestinal disease affecting many mammals including humans. Typically, in healthy individuals, cryptosporidiosis is a self-limiting disease. However, C. parvum can cause a severe and persistent infection that can be life-threatening for immunocompromised individuals, such as AIDS patients. As there are no available treatments for these patients that can cure the disease, there is an urgent need to identify treatment options. We tested the anti-parasitic activity of the alkylphosphocholine oleylphosphocholine (OlPC), an analog of miltefosine, against C. parvum in in vitro and in vivo studies. In vitro experiments using C. parvum infected human ileocecal adenocarcinoma cells (HCT-8 cells) showed that OlPC has an EC50 of 18.84 nM. Moreover, no cell toxicity has been seen at concentrations ≤50 μM. C57BL/6 interferon gamma receptor knock-out mice, were infected by gavage with 4000 C. parvum oocysts on Day 0. Oral treatments, with OlPC, miltefosine, paromomycin or PBS, began on Day 3 post-infection for 10 days. Treatment with OlPC, at 40 mg/kg/day resulted in 100% survival, complete clearance of parasite in stools and a 99.9% parasite burden reduction in the intestines at Day 30. Doses of 30 and 20 mg/kg/day also demonstrated an increased survival rate and a dose-dependent parasite burden reduction. Mice treated with 10 mg/kg/day of miltefosine resulted in 50% survival at Day 30. In contrast, control mice, treated with PBS or 100 mg/kg/day of paromomycin, died or had to be euthanized between Days 6 and 13 due to severe illness. Results of parasite burden were obtained by qPCR and cross-validated by both flow cytometry of stool oocysts and histological sections of the ileum. Together, our results strongly support that OlPC represents a potential candidate for the treatment of C. parvum infections in immunocompromised patients. PMID:26441906

  20. Oleylphosphocholine (OlPC) arrests Cryptosporidium parvum growth in vitro and prevents lethal infection in interferon gamma receptor knock-out mice.

    PubMed

    Sonzogni-Desautels, Karine; Renteria, Axel E; Camargo, Fabio V; Di Lenardo, Thomas Z; Mikhail, Alexandre; Arrowood, Michael J; Fortin, Anny; Ndao, Momar

    2015-01-01

    Cryptosporidium parvum is a species of protozoa that causes cryptosporidiosis, an intestinal disease affecting many mammals including humans. Typically, in healthy individuals, cryptosporidiosis is a self-limiting disease. However, C. parvum can cause a severe and persistent infection that can be life-threatening for immunocompromised individuals, such as AIDS patients. As there are no available treatments for these patients that can cure the disease, there is an urgent need to identify treatment options. We tested the anti-parasitic activity of the alkylphosphocholine oleylphosphocholine (OlPC), an analog of miltefosine, against C. parvum in in vitro and in vivo studies. In vitro experiments using C. parvum infected human ileocecal adenocarcinoma cells (HCT-8 cells) showed that OlPC has an EC50 of 18.84 nM. Moreover, no cell toxicity has been seen at concentrations ≤50 μM. C57BL/6 interferon gamma receptor knock-out mice, were infected by gavage with 4000 C. parvum oocysts on Day 0. Oral treatments, with OlPC, miltefosine, paromomycin or PBS, began on Day 3 post-infection for 10 days. Treatment with OlPC, at 40 mg/kg/day resulted in 100% survival, complete clearance of parasite in stools and a 99.9% parasite burden reduction in the intestines at Day 30. Doses of 30 and 20 mg/kg/day also demonstrated an increased survival rate and a dose-dependent parasite burden reduction. Mice treated with 10 mg/kg/day of miltefosine resulted in 50% survival at Day 30. In contrast, control mice, treated with PBS or 100 mg/kg/day of paromomycin, died or had to be euthanized between Days 6 and 13 due to severe illness. Results of parasite burden were obtained by qPCR and cross-validated by both flow cytometry of stool oocysts and histological sections of the ileum. Together, our results strongly support that OlPC represents a potential candidate for the treatment of C. parvum infections in immunocompromised patients. PMID:26441906

  1. Dipicrylamine Modulates GABAρ1 Receptors through Interactions with Residues in the TM4 and Cys-Loop Domains.

    PubMed

    Limon, Agenor; Estrada-Mondragón, Argel; Ruiz, Jorge M Reyes; Miledi, Ricardo

    2016-04-01

    Dipicrylamine (DPA) is a commonly used acceptor agent in Förster resonance energy transfer experiments that allows the study of high-frequency neuronal activity in the optical monitoring of voltage in living cells. However, DPA potently antagonizes GABAA receptors that contain α1 and β2 subunits by a mechanism which is not clearly understood. In this work, we aimed to determine whether DPA modulation is a general phenomenon of Cys-loop ligand-gated ion channels (LGICs), and whether this modulation depends on particular amino acid residues. For this, we studied the effects of DPA on human homomeric GABAρ1, α7 nicotinic, and 5-HT3A serotonin receptors expressed in Xenopus oocytes. Our results indicate that DPA is an allosteric modulator of GABAρ1 receptors with an IC50 of 1.6 µM, an enhancer of α7 nicotinic receptors at relatively high concentrations of DPA, and has little, if any, effect on 5-HT3A receptors. DPA antagonism of GABAρ1 was strongly enhanced by preincubation, was slightly voltage-dependent, and its washout was accelerated by bovine serum albumin. These results indicate that DPA modulation is not a general phenomenon of LGICs, and structural differences between receptors may account for disparities in DPA effects. In silico modeling of DPA docking to GABAρ1, α7 nicotinic, and 5-HT3A receptors suggests that a hydrophobic pocket within the Cys-loop and the M4 segment in GABAρ1, located at the extracellular/membrane interface, facilitates the interaction with DPA that leads to inhibition of the receptor. Functional examinations of mutant receptors support the involvement of the M4 segment in the allosteric modulation of GABAρ1 by DPA. PMID:26869399

  2. The Role of 5-HT3 Receptors in Signaling from Taste Buds to Nerves.

    PubMed

    Larson, Eric D; Vandenbeuch, Aurelie; Voigt, Anja; Meyerhof, Wolfgang; Kinnamon, Sue C; Finger, Thomas E

    2015-12-01

    Activation of taste buds triggers the release of several neurotransmitters, including ATP and serotonin (5-hydroxytryptamine; 5-HT). Type III taste cells release 5-HT directly in response to acidic (sour) stimuli and indirectly in response to bitter and sweet tasting stimuli. Although ATP is necessary for activation of nerve fibers for all taste stimuli, the role of 5-HT is unclear. We investigated whether gustatory afferents express functional 5-HT3 receptors and, if so, whether these receptors play a role in transmission of taste information from taste buds to nerves. In mice expressing GFP under the control of the 5-HT(3A) promoter, a subset of cells in the geniculate ganglion and nerve fibers in taste buds are GFP-positive. RT-PCR and in situ hybridization confirmed the presence of 5-HT(3A) mRNA in the geniculate ganglion. Functional studies show that only those geniculate ganglion cells expressing 5-HT3A-driven GFP respond to 10 μM 5-HT and this response is blocked by 1 μM ondansetron, a 5-HT3 antagonist, and mimicked by application of 10 μM m-chlorophenylbiguanide, a 5-HT3 agonist. Pharmacological blockade of 5-HT3 receptors in vivo or genetic deletion of the 5-HT3 receptors reduces taste nerve responses to acids and other taste stimuli compared with controls, but only when urethane was used as the anesthetic. We find that anesthetic levels of pentobarbital reduce taste nerve responses apparently by blocking the 5-HT3 receptors. Our results suggest that 5-HT released from type III cells activates gustatory nerve fibers via 5-HT3 receptors, accounting for a significant proportion of the neural taste response.

  3. Impact of intracellular domain flexibility upon properties of activated human 5-HT3 receptors*

    PubMed Central

    Kozuska, J L; Paulsen, I M; Belfield, W J; Martin, I L; Cole, D J; Holt, A; Dunn, S M J

    2014-01-01

    Background and Purpose It has been proposed that arginine residues lining the intracellular portals of the homomeric 5-HT3A receptor cause electrostatic repulsion of cation flow, accounting for a single-channel conductance substantially lower than that of the 5-HT3AB heteromer. However, comparison of receptor homology models for wild-type pentamers suggests that salt bridges in the intracellular domain of the homomer may impart structural rigidity, and we hypothesized that this rigidity could account for the low conductance. Experimental Approach Mutations were introduced into the portal region of the human 5-HT3A homopentamer, such that putative salt bridges were broken by neutralizing anionic partners. Single-channel and whole cell currents were measured in transfected tsA201 cells and in Xenopus oocytes respectively. Computational simulations of protein flexibility facilitated comparison of wild-type and mutant receptors. Key Results Single-channel conductance was increased substantially, often to wild-type heteromeric receptor values, in most 5-HT3A mutants. Conversely, introduction of arginine residues to the portal region of the heteromer, conjecturally creating salt bridges, decreased conductance. Gating kinetics varied significantly between different mutant receptors. EC50 values for whole-cell responses to 5-HT remained largely unchanged, but Hill coefficients for responses to 5-HT were usually significantly smaller in mutants. Computational simulations suggested increased flexibility throughout the protein structure as a consequence of mutations in the intracellular domain. Conclusions and Implications These data support a role for intracellular salt bridges in maintaining the quaternary structure of the 5-HT3 receptor and suggest a role for the intracellular domain in allosteric modulation of cooperativity and agonist efficacy. Linked Article This article is commented on by Vardy and Kenakin, pp. 1614–1616 of volume 171 issue 7. To view this commentary

  4. The Role of 5-HT3 Receptors in Signaling from Taste Buds to Nerves.

    PubMed

    Larson, Eric D; Vandenbeuch, Aurelie; Voigt, Anja; Meyerhof, Wolfgang; Kinnamon, Sue C; Finger, Thomas E

    2015-12-01

    Activation of taste buds triggers the release of several neurotransmitters, including ATP and serotonin (5-hydroxytryptamine; 5-HT). Type III taste cells release 5-HT directly in response to acidic (sour) stimuli and indirectly in response to bitter and sweet tasting stimuli. Although ATP is necessary for activation of nerve fibers for all taste stimuli, the role of 5-HT is unclear. We investigated whether gustatory afferents express functional 5-HT3 receptors and, if so, whether these receptors play a role in transmission of taste information from taste buds to nerves. In mice expressing GFP under the control of the 5-HT(3A) promoter, a subset of cells in the geniculate ganglion and nerve fibers in taste buds are GFP-positive. RT-PCR and in situ hybridization confirmed the presence of 5-HT(3A) mRNA in the geniculate ganglion. Functional studies show that only those geniculate ganglion cells expressing 5-HT3A-driven GFP respond to 10 μM 5-HT and this response is blocked by 1 μM ondansetron, a 5-HT3 antagonist, and mimicked by application of 10 μM m-chlorophenylbiguanide, a 5-HT3 agonist. Pharmacological blockade of 5-HT3 receptors in vivo or genetic deletion of the 5-HT3 receptors reduces taste nerve responses to acids and other taste stimuli compared with controls, but only when urethane was used as the anesthetic. We find that anesthetic levels of pentobarbital reduce taste nerve responses apparently by blocking the 5-HT3 receptors. Our results suggest that 5-HT released from type III cells activates gustatory nerve fibers via 5-HT3 receptors, accounting for a significant proportion of the neural taste response. PMID:26631478

  5. Positron emission tomographic imaging of the cannabinoid type 1 receptor system with [¹¹C]OMAR ([¹¹C]JHU75528): improvements in image quantification using wild-type and knockout mice.

    PubMed

    Herance, Raúl; Rojas, Santiago; Abad, Sergio; Jiménez, Xavier; Gispert, Juan Domingo; Millán, Olga; Martín-García, Elena; Burokas, Aurelijus; Serra, Miquel Àngel; Maldonado, Rafael; Pareto, Deborah

    2011-12-01

    In this study, we assessed the feasibility of using positron emission tomography (PET) and the tracer [¹¹C]OMAR ([¹¹C]JHU75528), an analogue of rimonabant, to study the brain cannabinoid type 1 (CB1) receptor system. Wild-type (WT) and CB1 knockout (KO) animals were imaged at baseline and after pretreatment with blocking doses of rimonabant. Brain uptake in WT animals was higher (50%) than in KO animals in baseline conditions. After pretreatment with rimonabant, WT uptake lowered to the level of KO animals. The results of this study support the feasibility of using PET with the radiotracer [¹¹C]JHU75528 to image the brain CB1 receptor system in mice. In addition, this methodology can be used to assess the effect of new drugs in preclinical studies using genetically manipulated animals.

  6. Blockade of 5-HT1A receptors by (+/-)-pindolol potentiates cortical 5-HT outflow, but not antidepressant-like activity of paroxetine: microdialysis and behavioral approaches in 5-HT1A receptor knockout mice.

    PubMed

    Guilloux, Jean-Philippe; David, Denis J P; Guiard, Bruno P; Chenu, Franck; Repérant, Christelle; Toth, Miklos; Bourin, Michel; Gardier, Alain M

    2006-10-01

    Selective serotonin reuptake inhibitors like paroxetine (Prx) often requires 4-6 weeks to achieve clinical benefits in depressed patients. Pindolol shortens this delay and it has been suggested that this effect is mediated by somatodendritic 5-hydroxytryptamine (5-HT) 1A autoreceptors. However clinical data on the beneficial effects of pindolol are conflicting. To study the effects of (+/-)-pindolol-paroxetine administration, we used genetical and pharmacological approaches in 5-HT1A knockout mice (5-HT1A-/-). Two assays, in vivo intracerebral microdialysis in awake mice and the forced swimming test (FST), were used to assess the antidepressant-like effects of this drug combination. Basal levels of extracellular serotonin, 5-HT ([5-HT]ext) in the frontal cortex (FCX) and the dorsal raphe nucleus (DRN) did not differ between the two strains of mice, suggesting a lack of tonic control of 5-HT1A autoreceptors on nerve terminal 5-HT release. Prx (1 and 4 mg/kg) dose-dependently increased cortical [5-HT]ext in both genotypes, but the effects were greater in mutants. The selective 5-HT1A receptor antagonist, WAY-100635 (0.5 mg/kg), or (+/-)-pindolol (5 and 10 mg/kg) potentiated the effects of Prx (4 mg/kg) on cortical [5-HT]ext in 5-HT1A+/+, but not in 5-HT1A-/- mice. Similar responses were obtained following local intra-raphe perfusion by reverse microdialysis of either WAY-100635 or (+/-)-pindolol (100 microM each). In the FST, Prx administration dose-dependently decreased the immobility time in both strains of mice, but the response was much greater in 5HT1A-/- mice. In contrast, (+/-)-pindolol blocked Prx-induced decreases in the immobility time while WAY-100635 had no effect in both genotypes. These findings using 5-HT1A-/- mice confirm that (+/-)-pindolol behaves as an antagonist of 5-HT1A autoreceptor in mice, but its blockade of paroxetine-induced antidepressant-like effects in the FST may be due to its binding to other neurotransmitter receptors.

  7. Expression of 5-HT3 receptors and TTX resistant sodium channels (NaV1.8) on muscle nerve fibers in pain-free humans and patients with chronic myofascial temporomandibular disorders

    PubMed Central

    2014-01-01

    Background Previous studies have shown that 5-HT3-antagonists reduce muscle pain, but there are no studies that have investigated the expression of 5-HT3-receptors in human muscles. Also, tetrodotoxin resistant voltage gated sodium-channels (NaV) are involved in peripheral sensitization and found in trigeminal ganglion neurons innervating the rat masseter muscle. This study aimed to investigate the frequency of nerve fibers that express 5-HT3A-receptors alone and in combination with NaV1.8 sodium-channels in human muscles and to compare it between healthy pain-free men and women, the pain-free masseter and tibialis anterior muscles, and patients with myofascial temporomandibular disorders (TMD) and pain-free controls. Methods Three microbiopsies were obtained from the most bulky part of the tibialis and masseter muscles of seven and six healthy men and seven and six age-matched healthy women, respectively, while traditional open biopsies were obtained from the most painful spot of the masseter of five female patients and from a similar region of the masseter muscle of five healthy, age-matched women. The biopsies were processed by routine immunohistochemical methods. The biopsy sections were incubated with monoclonal antibodies against the specific axonal marker PGP 9.5, and polyclonal antibodies against the 5-HT3A-receptors and NaV1.8 sodium-channels. Results A similar percentage of nerve fibers in the healthy masseter (85.2%) and tibialis (88.7%) muscles expressed 5-HT3A-receptors. The expression of NaV1.8 by 5-HT3A positive nerve fibers associated with connective tissue was significantly higher than nerve fibers associated with myocytes (P < .001). In the patients, significantly more fibers per section were found with an average of 3.8 ± 3 fibers per section in the masseter muscle compared to 2.7 ± 0.2 in the healthy controls (P = .024). Further, the frequency of nerve fibers that co-expressed NaV1.8 and 5-HT3A receptors was significantly

  8. Corynebacterium parvum- and Mycobacterium bovis bacillus Calmette-Guerin-induced granuloma formation is inhibited in TNF receptor I (TNF-RI) knockout mice and by treatment with soluble TNF-RI.

    PubMed

    Senaldi, G; Yin, S; Shaklee, C L; Piguet, P F; Mak, T W; Ulich, T R

    1996-12-01

    The aim of this study was to examine the role of TNF receptor I (TNF-RI) in the pathogenesis of heat-killed Corynebacterium parvum- and live bacillus Calmette-Guerin (BCG)-induced granulomas. Granuloma formation was analyzed in TNF-RI knockout mice and after treatment with soluble TNF-RI (sTNF-RI). TNF-RI knockout mice injected with C. parvum or BCG developed fewer and smaller granulomas than wild-type control mice. Mice treated with sTNF-RI from days 7 to 13 after injection of C. parvum or BCG developed fewer and smaller granulomas than saline-treated control mice. Established granulomas regressed in rats treated with sTNF-RI from days 10 to 13 after injection of C. parvum. In conclusion, TNF signaling via TNF-RI contributes to the pathogenesis of C. parvum- and BCG-induced granulomas. sTNF-RI inhibits the development of granulomas and can cause the regression of established granulomas. PMID:8943410

  9. The use of knock-out mice unravels distinct roles for mGlu2 and mGlu3 metabotropic glutamate receptors in mechanisms of neurodegeneration/neuroprotection.

    PubMed

    Corti, Corrado; Battaglia, Giuseppe; Molinaro, Gemma; Riozzi, Barbara; Pittaluga, Anna; Corsi, Mauro; Mugnaini, Manolo; Nicoletti, Ferdinando; Bruno, Valeria

    2007-08-01

    Dual metabotropic glutamate 2/3 (mGlu2/3) receptor agonists have been examined with success in the clinic with positive proof of efficacy in several tests of anxiety and schizophrenia. Moreover, a large body of evidence has accumulated that these drugs have significant neuroprotective potential. An important discussion in the field deals with dissecting effects on mGlu2 versus effects on mGlu3 receptors, which is relevant for the potential use of subtype-selective agonists or allosteric activators. We addressed this issue using mGlu2 and mGlu3 receptor knock-out mice. We used mixed cultures of cortical cells in which astrocytes and neurons were plated at different times and could therefore originate from different mice. Cultures were challenged with NMDA for the induction of excitotoxic neuronal death. The mGlu2/3 receptor agonist, (-)-2-oxa-4-aminocyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY379268), was equally neuroprotective in cultures containing neurons from wild-type, mGlu2-/-, or mGlu3-/- mice. Neuroprotection was instead abolished when astrocytes lacked mGlu3 receptors, unless neuronal mGlu2 receptors were also absent. The latter condition partially restored the protective activity of LY379268. Cultures in which neurons originated from mGlu2-/- mice were also intrinsically resistant to NMDA toxicity. In in vivo experiments, systemic administration of LY379268 protected striatal neurons against NMDA toxicity in wild-type and mGlu2-/- mice but not in mGlu3-/- mice. In addition, LY379268 was protective against nigrostriatal degeneration induced by low doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine only in mice lacking mGlu2 receptors. We conclude that neuroprotection by mGlu2/3 receptor agonists requires the activation of astrocytic mGlu3 receptors, whereas, unexpectedly, activation of mGlu2 receptors might be harmful to neurons exposed to toxic insults. PMID:17670976

  10. In vivo SPECT and ex vivo autoradiographic brain imaging of the novel selective CB1 receptor antagonist radioligand [125I]SD7015 in CB1 knock-out and wildtype mouse

    PubMed Central

    Máthé, Domokos; Horváth, Ildikó; Szigeti, Krisztián; Donohue, Sean R.; Pike, Victor W.; Jia, Zisheng; Ledent, Catherine; Palkovits, Miklós; Freund, Tamás F.; Halldin, Christer; Gulyás, Balázs

    2014-01-01

    We aimed to evaluate the novel high-affinity and relatively lipophilic CB1 receptor (CB1R) antagonist radioligand [125I]SD7015 for SPECT imaging of CB1Rs in vivo using the multiplexed multipinhole dedicated small animal SPECT/CT system, NanoSPECT/CTPLUS (Mediso, Budapest, Hungary), in knock-out CB1 receptor knock-out (CB1R-/-) and wildtype mice. In order to exclude possible differences in cerebral blood flow between the two types of animals, HMPAO SPECT scans were performed, whereas in order to confirm the brain uptake differences of the radioligand between knock-out mice and wildtype mice, in vivo scans were complemented with ex vivo autoradiographic measurements using the brains of the same animals. With SPECT/CT imaging, we measured the brain uptake of radioactivity, using %SUV (% standardised uptake values) in CB1R-/- mice (n = 3) and C57BL6 wildtype mice (n = 7) under urethane anaesthesia after injecting [125I]SD7015 intravenously or intraperitoneally. The Brookhaven Laboratory mouse MRI atlas was fused to the SPECT/CT images by using a combination of rigid and non-rigid algorithms in the Mediso Fusion™ (Mediso, Budapest, Hungary) and VivoQuant (inviCRO, Boston, MA, USA) softwares. Phosphor imager plate autoradiography (ARG) was performed on 4 μm-thin cryostat sections of the excised brains. %SUV was 8.6 ± 3.6 (average ± SD) in CB1R-/- mice and 22.1 ± 12.4 in wildtype mice between 2 and 4 h after injection (p < 0.05). ARG of identically taken sections from wildtype mouse brain showed moderate radioactivity uptake when compared with the in vivo images, with a clear difference between grey matter and white matter, whereas ARG in CB1R(-/-) mice showed practically no radioactivity uptake. [125I]SD7015 enters the mouse brain in sufficient amount to enable SPECT imaging. Brain radioactivity distribution largely coincides with that of the known CB1R expression pattern in rodent brain. We conclude that [125I]SD7015 should be a useful SPECT radioligand for

  11. In vivo SPECT and ex vivo autoradiographic brain imaging of the novel selective CB1 receptor antagonist radioligand [125I]SD7015 in CB1 knock-out and wildtype mouse.

    PubMed

    Máthé, Domokos; Horváth, Ildikó; Szigeti, Krisztián; Donohue, Sean R; Pike, Victor W; Jia, Zisheng; Ledent, Catherine; Palkovits, Miklós; Freund, Tamás F; Halldin, Christer; Gulyás, Balázs

    2013-02-01

    We aimed to evaluate the novel high-affinity and relatively lipophilic CB(1) receptor (CB(1)R) antagonist radioligand [(125)I]SD7015 for SPECT imaging of CB(1)Rs in vivo using the multiplexed multipinhole dedicated small animal SPECT/CT system, NanoSPECT/CT(PLUS) (Mediso, Budapest, Hungary), in knock-out CB(1) receptor knock-out (CB(1)R-/-) and wildtype mice. In order to exclude possible differences in cerebral blood flow between the two types of animals, HMPAO SPECT scans were performed, whereas in order to confirm the brain uptake differences of the radioligand between knock-out mice and wildtype mice, in vivo scans were complemented with ex vivo autoradiographic measurements using the brains of the same animals. With SPECT/CT imaging, we measured the brain uptake of radioactivity, using %SUV (% standardised uptake values) in CB(1)R-/- mice (n=3) and C57BL6 wildtype mice (n=7) under urethane anaesthesia after injecting [(125)I]SD7015 intravenously or intraperitoneally. The Brookhaven Laboratory mouse MRI atlas was fused to the SPECT/CT images by using a combination of rigid and non-rigid algorithms in the Mediso Fusion™ (Mediso, Budapest, Hungary) and VivoQuant (inviCRO, Boston, MA, USA) softwares. Phosphor imager plate autoradiography (ARG) was performed on 4 μm-thin cryostat sections of the excised brains. %SUV was 8.6±3.6 (average±SD) in CB(1)R-/- mice and 22.1±12.4 in wildtype mice between 2 and 4 h after injection (p<0.05). ARG of identically taken sections from wildtype mouse brain showed moderate radioactivity uptake when compared with the in vivo images, with a clear difference between grey matter and white matter, whereas ARG in CB(1)R(-/-) mice showed practically no radioactivity uptake. [(125)I]SD7015 enters the mouse brain in sufficient amount to enable SPECT imaging. Brain radioactivity distribution largely coincides with that of the known CB(1)R expression pattern in rodent brain. We conclude that [(125)I]SD7015 should be a useful SPECT

  12. mRNA transfection of a novel TAL effector nuclease (TALEN) facilitates efficient knockout of HIV co-receptor CCR5.

    PubMed

    Mock, Ulrike; Machowicz, Rafał; Hauber, Ilona; Horn, Stefan; Abramowski, Pierre; Berdien, Belinda; Hauber, Joachim; Fehse, Boris

    2015-06-23

    Homozygosity for a natural deletion variant of the HIV-coreceptor molecule CCR5, CCR5Δ32, confers resistance toward HIV infection. Allogeneic stem cell transplantation from a CCR5Δ32-homozygous donor has resulted in the first cure from HIV ('Berlin patient'). Based thereon, genetic disruption of CCR5 using designer nucleases was proposed as a promising HIV gene-therapy approach. Here we introduce a novel TAL-effector nuclease, CCR5-Uco-TALEN that can be efficiently delivered into T cells by mRNA electroporation, a gentle and truly transient gene-transfer technique. CCR5-Uco-TALEN mediated high-rate CCR5 knockout (>90% in PM1 and >50% in primary T cells) combined with low off-target activity, as assessed by flow cytometry, next-generation sequencing and a newly devised, very convenient gene-editing frequency digital-PCR (GEF-dPCR). GEF-dPCR facilitates simultaneous detection of wild-type and gene-edited alleles with remarkable sensitivity and accuracy as shown for the CCR5 on-target and CCR2 off-target loci. CCR5-edited cells were protected from infection with HIV-derived lentiviral vectors, but also with the wild-type CCR5-tropic HIV-1BaL strain. Long-term exposure to HIV-1BaL resulted in almost complete suppression of viral replication and selection of CCR5-gene edited T cells. In conclusion, we have developed a novel TALEN for the targeted, high-efficiency knockout of CCR5 and a useful dPCR-based gene-editing detection method.

  13. GABA-A receptor inhibition of local calcium signaling in spines and dendrites.

    PubMed

    Marlin, Joseph J; Carter, Adam G

    2014-11-26

    Cortical interneurons activate GABA-A receptors to rapidly control electrical and biochemical signaling at pyramidal neurons. Different populations of interneurons are known to uniquely target the soma and dendrites of pyramidal neurons. However, the ability of these interneurons to inhibit Ca(2+) signaling at spines and dendrites is largely unexplored. Here we use whole-cell recordings, two-photon microscopy, GABA uncaging and optogenetics to study dendritic inhibition at layer 5 (L5) pyramidal neurons in slices of mouse PFC. We first show that GABA-A receptors strongly inhibit action potential (AP)-evoked Ca(2+) signals at both spines and dendrites. We find robust inhibition over tens of milliseconds that spreads along the dendritic branch. However, we observe no difference in the amount of inhibition at neighboring spines and dendrites. We then examine the influence of interneurons expressing parvalbumin (PV), somatostatin (SOM), or 5HT3a receptors. We determine that these populations of interneurons make unique contacts onto the apical and basal dendrites of L5 pyramidal neurons. We also show that SOM and 5HT3a but not PV interneurons potently inhibit AP Ca(2+) signals via GABA-A receptors at both spines and dendrites. These findings reveal how multiple interneurons regulate local Ca(2+) signaling in pyramidal neurons, with implications for cortical function and disease.

  14. Specific Activation of A3, A2A and A1 Adenosine Receptors in CD73-Knockout Mice Affects B16F10 Melanoma Growth, Neovascularization, Angiogenesis and Macrophage Infiltration

    PubMed Central

    Koszałka, Patrycja; Gołuńska, Monika; Urban, Aleksandra; Stasiłojć, Grzegorz; Stanisławowski, Marcin; Majewski, Marceli; Składanowski, Andrzej C.; Bigda, Jacek

    2016-01-01

    CD73 (ecto-5'-nucleotidase), a cell surface enzyme hydrolyzing AMP to adenosine, was lately demonstrated to play a direct role in tumor progression including regulation of tumor vascularization. It was also shown to stimulate tumor macrophage infiltration. Interstitial adenosine, accumulating in solid tumors due to CD73 enzymatic activity, is recognized as a main mediator regulating the production of pro- and anti-angiogenic factors, but the engagement of specific adenosine receptors in tumor progression in vivo is still poorly researched. We have analyzed the role of high affinity adenosine receptors A1, A2A, and A3 in B16F10 melanoma progression using specific agonists (CCPA, CGS-21680 and IB-MECA, respectively). We limited endogenous extracellular adenosine background using CD73 knockout mice treated with CD73 chemical inhibitor, AOPCP (adenosine α,β-methylene 5’-diphosphate). Activation of any adenosine receptor significantly inhibited B16F10 melanoma growth but only at its early stage. At 14th day of growth, the decrease in tumor neovascularization and MAPK pathway activation induced by CD73 depletion was reversed by all agonists. Activation of A1AR primarily increased angiogenic activation measured by expression of VEGF-R2 on tumor blood vessels. However, mainly A3AR activation increased both the microvessel density and expression of pro-angiogenic factors. All agonists induced significant increase in macrophage tumor infiltration, with IB-MECA being most effective. This effect was accompanied by substantial changes in cytokines regulating macrophage polarization between pro-inflammatory and pro-angiogenic phenotype. Our results demonstrate an evidence that each of the analyzed receptors has a specific role in the stimulation of tumor angiogenesis and confirm significantly more multifaceted role of adenosine in its regulation than was already observed. They also reveal previously unexplored consequences to extracellular adenosine signaling depletion in

  15. Combined behavioral studies and in vivo imaging of inflammatory response and expression of mGlu5 receptors in schnurri-2 knockout mice.

    PubMed

    Choi, Ji-Kyung; Zhu, Aijun; Jenkins, Bruce G; Hattori, Satoko; Kil, Kun-Eek; Takagi, Tsuyoshi; Ishii, Shunsuke; Miyakawa, Tsuyoshi; Brownell, Anna-Liisa

    2015-11-16

    Schnurri-2 (Shn-2) knockout (KO) mice have been proposed as a preclinical neuroinflammatory schizophrenia model. We used behavioral studies and imaging markers that can be readily translated to human populations to explore brain effects of inflammation. Shn-2 KO mice and their littermate control mice were imaged with two novel PET ligands; an inflammation marker [(11)C]PBR28 and the mGluR5 ligand [(18)F]FPEB. Locomotor activity was measured using open field exploration with saline, methamphetamine or amphetamine challenge. A significantly increased accumulation of [(11)C]PBR28 was found in the cortex, striatum, hippocampus and olfactory bulb of Shn-2 KO mice. Increased mGluR5 binding was also observed in the cortex and hippocampus of the Shn-2 KO mice. Open field locomotor testing revealed a large increase in novelty-induced hyperlocomotion in Shn-2 KO mice with abnormal (decreased) responses to either methamphetamine or amphetamine. These data provide additional support to demonstrate that the Shn-2 KO mouse model exhibits several behavioral and pathological markers resembling human schizophrenia making it an attractive translational model for the disease.

  16. A cluster of novel serotonin receptor 3-like genes on human chromosome 3.

    PubMed

    Karnovsky, Alla M; Gotow, Lisa F; McKinley, Denise D; Piechan, Julie L; Ruble, Cara L; Mills, Cynthia J; Schellin, Kathleen A B; Slightom, Jerry L; Fitzgerald, Laura R; Benjamin, Christopher W; Roberds, Steven L

    2003-11-13

    The ligand-gated ion channel family includes receptors for serotonin (5-hydroxytryptamine, 5-HT), acetylcholine, GABA, and glutamate. Drugs targeting subtypes of these receptors have proven useful for the treatment of various neuropsychiatric and neurological disorders. To identify new ligand-gated ion channels as potential therapeutic targets, drafts of human genome sequence were interrogated. Portions of four novel genes homologous to 5-HT(3A) and 5-HT(3B) receptors were identified within human sequence databases. We named the genes 5-HT(3C1)-5-HT(3C4). Radiation hybrid (RH) mapping localized these genes to chromosome 3q27-28. All four genes shared similar intron-exon organizations and predicted protein secondary structure with 5-HT(3A) and 5-HT(3B). Orthologous genes were detected by Southern blotting in several species including dog, cow, and chicken, but not in rodents, suggesting that these novel genes are not present in rodents or are very poorly conserved. Two of the novel genes are predicted to be pseudogenes, but two other genes are transcribed and spliced to form appropriate open reading frames. The 5-HT(3C1) transcript is expressed almost exclusively in small intestine and colon, suggesting a possible role in the serotonin-responsiveness of the gut.

  17. Accumulation of cytolytic CD8{sup +} T cells in B16-melanoma and proliferation of mature T cells in TIS21-knockout mice after T cell receptor stimulation

    SciTech Connect

    Ryu, Min Sook; Woo, Min-Yeong; Kwon, Daeho; Hong, Allen E.; Song, Kye Yong; Park, Sun; Lim, In Kyoung

    2014-10-01

    In vivo and in vitro effects of TIS21 gene on the mature T cell activation and antitumor activities were explored by employing MO5 melanoma orthograft and splenocytes isolated from the TIS21-knockout (KO) mice. Proliferation and survival of mature T cells were significantly increased in the KO than the wild type (WT) cells, indicating that TIS21 inhibits the rate of mature T cell proliferation and its survival. In MO5 melanoma orthograft model, the KO mice recruited much more CD8{sup +} T cells into the tumors at around day 14 after tumor cell injection along with reduced tumor volumes compared with the WT. The increased frequency of granzyme B{sup +} CD8{sup +} T cells in splenocytes of the KO mice compared with the WT may account for antitumor-immunity of TIS21 gene in the melanoma orthograft. In contrast, reduced frequencies of CD107a{sup +} CD8{sup +} T cells in the splenocytes of KO mice may affect the loss of CD8{sup +} T cell infiltration in the orthograft at around day 19. These results indicate that TIS21 exhibits antiproliferative and proapoptotic effects in mature T cells, and differentially affects the frequencies of granzyme B{sup +} CD8{sup +} T-cells and CD107a{sup +} CD8{sup +} T-cells, thus transiently regulating in vivo anti-tumor immunity. - Highlights: • Constitutive expression of TIS21 in splenocytes and upregulation by TCR stimulation. • Proliferation of mature T-cells in spleen of TIS21KO mice after TCR stimulation. • Inhibition of cell death in mature T-cells of TIS21KO mice compared with the wild type. • Inhibition of melanoma growth in TIS21KO mice and CD8{sup +} T cell infiltration in tumor. • Reduction of CD 107{sup +}CD8{sup +} T cells, but increased granzyme B{sup +} CD8{sup +} T cells in TIS21KO mice.

  18. The role of group II metabotropic glutamate receptors in cognition and anxiety: Comparative studies in GRM2−/−, GRM3−/− and GRM2/3−/− knockout mice

    PubMed Central

    De Filippis, Bianca; Lyon, Louisa; Taylor, Amy; Lane, Tracy; Burnet, Philip W.J.; Harrison, Paul J.; Bannerman, David M.

    2015-01-01

    Group II metabotropic glutamate receptors (mGlu2 and mGlu3, encoded by GRM2 and GRM3) have been implicated in both cognitive and emotional processes, although their precise role remains to be established. Studies with knockout (KO) mice provide an important approach for investigating the role of specific receptor genes in behaviour. In the present series of experiments we extended our prior characterisation of GRM2/3−/− double KO mice and, in complementary experiments, investigated the behavioural phenotype of single GRM2−/− and GRM3−/− mice. We found no consistent effect on anxiety in either the double or single KO mice. The lack of an anxiety phenotype in any of the lines contrasts with the clear anxiolytic effects of mGlu2/3 ligands. Motor co-ordination was impaired in GRM2/3−/− mice, but spared in single GRM2−/− and GRM3−/− mice. Spatial working memory (rewarded alternation) testing on the elevated T-maze revealed a deficit in GRM2−/− mice throughout testing, whereas GRM3−/− mice exhibited a biphasic effect (initially impaired, but performing better than controls by the end of training). A biphasic effect on activity levels was seen for the GRM2−/− mice. Overall, the phenotype in both GRM2−/− and GRM3−/− mice was less pronounced – if present at all – compared to GRM2/3−/− mice, across the range of task domains. This is consistent with possible redundancy of function and/or compensation in the single KO lines. Results are discussed with reference to a possible role for group II metabotropic glutamate receptors at the interface between arousal and behavioural performance, according to an inverted U-shaped function. PMID:25158312

  19. Analysis in conditional cannabinoid 1 receptor-knockout mice reveals neuronal subpopulation-specific effects on epileptogenesis in the kindling paradigm.

    PubMed

    von Rüden, E L; Jafari, M; Bogdanovic, R M; Wotjak, C T; Potschka, H

    2015-01-01

    The endocannabinoid system serves as a retrograde negative feedback mechanism. It is thought to control neuronal activity in an epileptic neuronal network. The purpose of this study was to evaluate the impact of the endocannabinoid and endovanilloid systems on both epileptogenesis and ictogenesis. Therefore, we modulated the endocannabinoid and endovanilloid systems genetically and pharmacologically, and analyzed the subsequent impact on seizure progression in the kindling model of temporal lobe epilepsy in mice. In addition, the impact of seizures on associated cellular alterations was evaluated. Our principal results revealed that the endocannabinoid system affects seizure and afterdischarge duration dependent on the neuronal subpopulation being modulated. Genetic deletion of CB1-receptors (CB1Rs) from principal neurons of the forebrain and pharmacological antagonism with rimonabant (5 mg/kg) caused longer seizure duration. Deletion of CB1R from GABAergic forebrain neurons resulted in the opposite effect. Along with these findings, the CB1R density was elevated in animals with repetitively induced seizures. However, neither genetic nor pharmacological interventions had any impact on the development of generalized seizures. Other than CB1, genetic deletion or pharmacological blockade with SB366791 (1 mg/kg) of transient receptor potential vanilloid receptor 1 (TRPV1) had no effect on the duration of behavioral or electrographic seizure activity in the kindling model. In conclusion, we demonstrate that endocannabinoid, but not endovanilloid, signaling affects termination of seizure activity, without influencing seizure severity over time. These effects are dependent on the neuronal subpopulation. Thus, the data argue that the endocannabinoid system plays an active role in seizure termination but does not regulate epileptogenesis.

  20. Alterations of gene expression of sodium channels in dorsal root ganglion neurons of estrogen receptor knockout (ERKO) mice induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).

    PubMed

    Ding, Haixia; Wang, Qiang; Liu, Jingli; Qian, Wenyi; Wang, Wenjuan; Wang, Jun; Gao, Rong; Xiao, Hang

    2012-08-01

    Estrogen receptors (ERα and ERβ) mediate the neuroprotection of estrogens against MPTP-induced striatal dopamine (DA) depletion. Pain is an important and distressing symptom in Parkinson's disease (PD). Voltage-gated sodium channels in sensory neurons are involved in the development of neuropathic pain. In this study, MPTP caused changes in nociception and alterations of gene expression of voltage-gated sodium channels in dorsal root ganglion (DRG) neurons in ER knockout (ERKO) mice were investigated. We found that administration of MPTP (11 mg/kg) to WT mice led to an extensive depletion of DA and its two metabolites, αERKO mice were observed to be more susceptible to MPTP toxicity than βERKO or WT mice. In addition, we found that the mRNA levels of TTX-S and TTX-R sodium channel subtypes were differentially affected in MPTP-treated WT animals. The MPTP-induced up-regulation of Nav1.1 and Nav1.9, down-regulation of Nav1.6 in DRG neurons may be through ERβ, up-regulation of Nav1.7 and down-regulation of Nav1.8 are dependent on both ERα and ERβ. Therefore, the MPTP-induced alterations of gene expression of sodium channels in DRG neurons could be an important mechanism to affect excitability and nociceptive thresholds, and the ERs appear to play a role in nociception in PD. PMID:22371119

  1. Fractionation of spatial memory in GRM2/3 (mGlu2/mGlu3) double knockout mice reveals a role for group II metabotropic glutamate receptors at the interface between arousal and cognition.

    PubMed

    Lyon, Louisa; Burnet, Philip W J; Kew, James N C; Corti, Corrado; Rawlins, J Nicholas P; Lane, Tracy; De Filippis, Bianca; Harrison, Paul J; Bannerman, David M

    2011-12-01

    Group II metabotropic glutamate receptors (mGluR2 and mGluR3, encoded by GRM2 and GRM3) are implicated in hippocampal function and cognition, and in the pathophysiology and treatment of schizophrenia and other psychiatric disorders. However, pharmacological and behavioral studies with group II mGluR agonists and antagonists have produced complex results. Here, we studied hippocampus-dependent memory in GRM2/3 double knockout (GRM2/3(-/-)) mice in an iterative sequence of experiments. We found that they were impaired on appetitively motivated spatial reference and working memory tasks, and on a spatial novelty preference task that relies on animals' exploratory drive, but were unimpaired on aversively motivated spatial memory paradigms. GRM2/3(-/-) mice also performed normally on an appetitively motivated, non-spatial, visual discrimination task. These results likely reflect an interaction between GRM2/3 genotype and the arousal-inducing properties of the experimental paradigm. The deficit seen on appetitive and exploratory spatial memory tasks may be absent in aversive tasks because the latter induce higher levels of arousal, which rescue spatial learning. Consistent with an altered arousal-cognition relationship in GRM2/3(-/-) mice, injection stress worsened appetitively motivated, spatial working memory in wild-types, but enhanced performance in GRM2/3(-/-) mice. GRM2/3(-/-) mice were also hypoactive in response to amphetamine. This fractionation of hippocampus-dependent memory depending on the appetitive-aversive context is to our knowledge unique, and suggests a role for group II mGluRs at the interface of arousal and cognition. These arousal-dependent effects may explain apparently conflicting data from previous studies, and have translational relevance for the involvement of these receptors in schizophrenia and other disorders. PMID:21832989

  2. Toll like receptor 2 knock-out attenuates carbon tetrachloride (CCl4)-induced liver fibrosis by downregulating MAPK and NF-κB signaling pathways.

    PubMed

    Ji, Lingling; Xue, Ruyi; Tang, Wenqing; Wu, Weibin; Hu, Tingting; Liu, Xijun; Peng, Xiaomin; Gu, Jianxin; Chen, She; Zhang, Si

    2014-06-01

    Innate immune signaling associated with Toll-like receptors (TLRs) is a key pathway involved in the progression of liver fibrosis. In this study, we reported that TLR2 is required for hepatic fibrogenesis induced by carbon tetrachloride (CCl4). After CCl4 treatment, TLR2(-/-) mice had reduced liver enzyme levels, diminished collagen deposition, decreased inflammatory infiltration and impaired activation of hepatic stellate cells (HSCs) than wild type (WT) mice. Furthermore, after CCl4 treatment, TLR2(-/-) mice demonstrated downregulated expression of profibrotic and proinflammatory genes and impaired mitogen-activated protein kinases (MAPK) and nuclear factor kappa B (NF-κB) activation than WT mice. Collectively, our data indicate that TLR2 deficiency protects against CCl4-induced liver fibrosis.

  3. TRAIL-Death Receptor 4 Signaling via Lysosome Fusion and Membrane Raft Clustering In Coronary Arterial Endothelial Cells: Evidence from ASM Knockout Mice

    PubMed Central

    Li, Xiang; Han, Wei-Qing; Boini, Krishna M.; Xia, Min; Zhang, Yang; Li, Pin-Lan

    2012-01-01

    Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) and its receptor death receptor 4 (DR4) have been implicated in the development of endothelial dysfunction and atherosclerosis. However, the signaling mechanism mediating DR4 activation and leading to endothelial injury remains unclear. We recently demonstrated that ceramide production via hydrolysis of membrane sphingomyelin by acid sphingomyelinase (ASM) results in membrane raft (MRs) clustering and formation of important redox signaling platforms, which play a crucial role in amplifying redox signaling in endothelial cells leading to endothelial dysfunction. The present study aims to investigate whether TRAIL triggers MR clustering via lysosome fusion and ASM activation, thereby conducting transmembrane redox signaling and changing endothelial function. Using confocal microscopy, we found that TRAIL induced MR clustering and its co-localization with DR4 in coronary arterial endothelial cells (CAECs) isolated from wild-type (Smpd1+/+) mice. Further, TRAIL triggered ASM translocation, ceramide production and NADPH oxidase aggregation in MR clusters in Smpd1+/+ CAECs, whereas these observations were not found in Smpd1−/− CAECs. Moreover, ASM deficiency reduced TRAIL-induced O2−· production in CAECs and abolished TRAIL-induced impairment on endothelium-dependent vasodilation in small resistance arteries. By measuring fluorescence resonance energy transfer (FRET), we found that Lamp-1 (lysosome membrane marker protein) and ganglioside GM1 (MR marker) were trafficking together in Smpd1+/+ CAECs, which was absent in Smpd1−/− CAECs. Consistently, fluorescence imaging of living cells with specific lysosome probes demonstrated that TRAIL-induced lysosome fusion with membrane was also absent in Smpd1−/− CAECs. Taken together, these results suggest that ASM is essential for TRAIL-induced lysosomal trafficking and fusion with membrane and formation of MR redox signaling platforms, which may

  4. CB1-receptor knockout neonatal mice are protected against ethanol-induced impairments of DNMT1, DNMT3A, and DNA methylation.

    PubMed

    Nagre, Nagaraja N; Subbanna, Shivakumar; Shivakumar, Madhu; Psychoyos, Delphine; Basavarajappa, Balapal S

    2015-02-01

    The significant consequences of ethanol use during pregnancy are neurobehavioral abnormalities involving hippocampal and neocortex malfunctions that cause learning and memory deficits collectively named fetal alcohol spectrum disorder. However, the molecular mechanisms underlying these abnormalities are still poorly understood and therefore warrant systematic research. Here, we document novel epigenetic abnormalities in the mouse model of fetal alcohol spectrum disorder. Ethanol treatment of P7 mice, which induces activation of caspase 3, impaired DNA methylation through reduced DNA methyltransferases (DNMT1 and DNMT3A) levels. Inhibition of caspase 3 activity, before ethanol treatment, rescued DNMT1, DNMT3A proteins as well as DNA methylation levels. Blockade of histone methyltransferase (G9a) activity or cannabinoid receptor type-1 (CB1R), prior to ethanol treatment, which, respectively, inhibits or prevents activation of caspase 3, rescued the DNMT1 and DNMT3A proteins and DNA methylation. No reduction of DNMT1 and DNMT3A proteins and DNA methylation was found in P7 CB1R null mice, which exhibit no ethanol-induced activation of caspase 3. Together, these data demonstrate that ethanol-induced activation of caspase 3 impairs DNA methylation through DNMT1 and DNMT3A in the neonatal mouse brain, and such impairments are absent in CB1R null mice. Epigenetic events mediated by DNA methylation may be one of the essential mechanisms of ethanol teratogenesis. Schematic mechanism of action by which ethanol impairs DNA methylation. Studies have demonstrated that ethanol has the capacity to bring epigenetic changes to contribute to the development of fetal alcohol spectrum disorder (FASD). However, the mechanisms are not well studied. P7 ethanol induces the activation of caspase 3 and impairs DNA methylation through reduced DNA methyltransferases (DNMT1 and DNMT3A) proteins (→). The inhibition or genetic ablation of cannabinoid receptor type-1 or inhibition of histone

  5. Double P2X2/P2X3 Purinergic Receptor Knockout Mice Do Not Taste NaCl or the Artificial Sweetener SC45647

    PubMed Central

    Eddy, Meghan C.; Eschle, Benjamin K.; Barrows, Jennell; Hallock, Robert M.; Finger, Thomas E.

    2009-01-01

    The P2X ionotropic purinergic receptors, P2X2 and P2X3, are essential for transmission of taste information from taste buds to the gustatory nerves. Mice lacking both P2X2 and P2X3 purinergic receptors (P2X2/P2X3Dbl−/−) exhibit no taste-evoked activity in the chorda tympani and glossopharyngeal nerves when stimulated with taste stimuli from any of the 5 classical taste quality groups (salt, sweet, sour, bitter, and umami) nor do the mice show taste preferences for sweet or umami, or avoidance of bitter substances (Finger et al. 2005. ATP signaling is crucial for communication from taste buds to gustatory nerves. Science. 310[5753]:1495–1499). Here, we compare the ability of P2X2/P2X3Dbl−/− mice and P2X2/P2X3Dbl+/+ wild-type (WT) mice to detect NaCl in brief-access tests and conditioned aversion paradigms. Brief-access testing with NaCl revealed that whereas WT mice decrease licking at 300 mM and above, the P2X2/P2X3Dbl−/− mice do not show any change in lick rates. In conditioned aversion tests, P2X2/P2X3Dbl−/− mice did not develop a learned aversion to NaCl or the artificial sweetener SC45647, both of which are easily avoided by conditioned WT mice. The inability of P2X2/P2X3Dbl−/− mice to show avoidance of these taste stimuli was not due to an inability to learn the task because both WT and P2X2/P2X3Dbl−/− mice learned to avoid a combination of SC45647 and amyl acetate (an odor cue). These data suggest that P2X2/P2X3Dbl−/− mice are unable to respond to NaCl or SC45647 as taste stimuli, mirroring the lack of gustatory nerve responses to these substances. PMID:19833661

  6. The Role of 5-HT3 Receptors in Signaling from Taste Buds to Nerves

    PubMed Central

    Vandenbeuch, Aurelie; Voigt, Anja; Meyerhof, Wolfgang; Kinnamon, Sue C.; Finger, Thomas E.

    2015-01-01

    Activation of taste buds triggers the release of several neurotransmitters, including ATP and serotonin (5-hydroxytryptamine; 5-HT). Type III taste cells release 5-HT directly in response to acidic (sour) stimuli and indirectly in response to bitter and sweet tasting stimuli. Although ATP is necessary for activation of nerve fibers for all taste stimuli, the role of 5-HT is unclear. We investigated whether gustatory afferents express functional 5-HT3 receptors and, if so, whether these receptors play a role in transmission of taste information from taste buds to nerves. In mice expressing GFP under the control of the 5-HT3A promoter, a subset of cells in the geniculate ganglion and nerve fibers in taste buds are GFP-positive. RT-PCR and in situ hybridization confirmed the presence of 5-HT3A mRNA in the geniculate ganglion. Functional studies show that only those geniculate ganglion cells expressing 5-HT3A-driven GFP respond to 10 μm 5-HT and this response is blocked by 1 μm ondansetron, a 5-HT3 antagonist, and mimicked by application of 10 μm m-chlorophenylbiguanide, a 5-HT3 agonist. Pharmacological blockade of 5-HT3 receptors in vivo or genetic deletion of the 5-HT3 receptors reduces taste nerve responses to acids and other taste stimuli compared with controls, but only when urethane was used as the anesthetic. We find that anesthetic levels of pentobarbital reduce taste nerve responses apparently by blocking the 5-HT3 receptors. Our results suggest that 5-HT released from type III cells activates gustatory nerve fibers via 5-HT3 receptors, accounting for a significant proportion of the neural taste response. SIGNIFICANCE STATEMENT Historically, serotonin (5-hydroxytryptamine; 5-HT) has been described as a candidate neurotransmitter in the gustatory system and recent studies show that type III taste receptor cells release 5-HT in response to various taste stimuli. In the present study, we demonstrate that a subset of gustatory sensory neurons express functional

  7. Activation of IKK/NF-κB provokes renal inflammatory responses in guanylyl cyclase/natriuretic peptide receptor-A gene-knockout mice

    PubMed Central

    Das, Subhankar; Periyasamy, Ramu

    2012-01-01

    The present study was aimed at determining the consequences of the disruption of guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) gene (Npr1) on proinflammatory responses of nuclear factor kappa B, inhibitory kappa B kinase, and inhibitory kappa B alpha (NF-κB, IKK, IκBα) in the kidneys of mutant mice. The results showed that the disruption of Npr1 enhanced the renal NF-κB binding activity by 3.8-fold in 0-copy (−/−) mice compared with 2-copy (+/+) mice. In parallel, IKK activity and IκBα protein phosphorylation were increased by 8- and 11-fold, respectively, in the kidneys of 0-copy mice compared with wild-type mice. Interestingly, IκBα was reduced by 80% and the expression of proinflammatory cytokines and renal fibrosis were significantly enhanced in 0-copy mice than 2-copy mice. Treatment of 0-copy mice with NF-κB inhibitors andrographolide, pyrrolidine dithiocarbamate, and etanercept showed a substantial reduction in renal fibrosis, attenuation of proinflammatory cytokines gene expression, and significantly reduced IKK activity and IkBα phosphorylation. These findings indicate that the systemic disruption of Npr1 activates the renal NF-κB pathways in 0-copy mice, which transactivates the expression of various proinflammatory cytokines to initiate renal remodeling; however, inhibition of NF-κB pathway repairs the abnormal renal pathology in mutant mice. PMID:22318993

  8. Effect of long-term ingestion of weakly oxidised flaxseed oil on biomarkers of oxidative stress in LDL-receptor knockout mice.

    PubMed

    Nogueira, M S; Kessuane, M C; Lobo Ladd, A A B; Lobo Ladd, F V; Cogliati, B; Castro, I A

    2016-07-01

    The effect of oxidised fatty acids on atherosclerosis progression is controversial. Thus, our objective was to evaluate the effect of long-term consumption of weakly oxidised PUFA from flaxseed oil on oxidative stress biomarkers of LDL-receptor(-/-) mice. To test our hypothesis, mice were separated into three groups. The first group received a high-fat diet containing fresh flaxseed oil (CONT-), the second was fed the same diet prepared using heated flaxseed oil (OXID), and the third group received the same diet containing fresh flaxseed oil and had diabetes induced by streptozotocin (CONT+). Oxidative stress, aortic parameters and non-alcoholic fatty liver disease were assessed. After 3 months, plasma lipid profile, glucose levels, body weight, energy intake and dietary intake did not differ among groups. Likewise, oxidative stress, plasma malondialdehyde (MDA), hepatic MDA expressed as nmol/mg portion (ptn) and antioxidant enzymes did not differ among the groups. Hepatic linoleic acid, α-linolenic acid, arachidonic acid and EPA acid declined in the OXID and CONT+ groups. Aortic wall thickness, lumen and diameter increased only in the OXID group. OXID and CONT+ groups exhibited higher concentrations of MDA, expressed as μmol/mg ptn per %PUFA, when compared with the CONT- group. Our results suggest that ingestion of oxidised flaxseed oil increases hepatic MDA concentration and is potentially pro-atherogenic. In addition, the mean MDA value observed in all groups was similar to those reported in other studies that used xenobiotics as oxidative stress inducers. Thus, the diet applied in this study represents an interesting model for further research involving antioxidants.

  9. Ultrasound Biomicroscopic Imaging for Interleukin-1 Receptor Antagonist-Inhibiting Atherosclerosis and Markers of Inflammation in Atherosclerotic Development in Apolipoprotein-E Knockout Mice.

    PubMed

    Li, Rong-Juan; Sun, Yan; Wang, Qin; Yang, Jiao; Yang, Ya; Song, Li; Wang, Zheng; Luo, Xiang-Hong; Su, Rui-Juan

    2015-08-01

    We sought to validate the hypothesis that the development of atherosclerosis can be suppressed by the interleukin-1 receptor antagonist (IL-1Ra) in murine models of atherosclerosis in vivo, noninvasively seen by means of high-resolution ultrasound biomicroscopy, and we studied changes in inflammatory markers such as IL-1 and C-reactive protein (CRP) plasma levels in these models of atherosclerosis. We divided IL-1Ra(+/-)/apolipoprotein-E (apoE)(-/-) and IL-1Ra(+/+)/apoE(-/-) mice into 2 age groups, used as atherosclerotic models. The control groups were age-matched IL-1Ra(+/+)/apoE(+/+) mice. Plaque thickness was measured in the ascending aorta in short-axis images by means of ultrasound and histology. Plasma levels of IL-1 and CRP were quantified in the 3 murine groups. At 16 weeks, plaque thickness in the ascending aortas of the IL-1Ra(+/-)/apoE(-/-) mice was significantly greater than that in the IL-1Ra(+/+)/apoE(-/-) mice, on ultrasound and histology (P <0.01). In contrast, at 32 weeks, the differences between these 2 genotypes were not statistically significant. Serum IL-1 levels were lower in the IL-1Ra(+/-)/apoE(-/-) mice than in the IL-1Ra(+/+)/apoE(-/-) mice at 16 and 32 weeks (P <0.05). At 16 weeks, serum CRP levels in the IL-1Ra(+/-)/apoE(-/-) mice were higher than in the IL-1Ra(+/+)/apoE(-/-) mice (P <0.01). Our results suggest that ultrasound biomicroscopy enables evaluation of atherosclerotic lesions in vivo, noninvasively and in real-time, in apoE(-/-) mice. Partial IL-1Ra deficiencies might promote early plaque development in 16-week-old apoE(-/-) mice. The balance of IL-1 and IL-1Ra might influence atherosclerotic development. Finally, CRP might affect the initiation of atherosclerosis, rather than its progression.

  10. Ultrasound Biomicroscopic Imaging for Interleukin-1 Receptor Antagonist-Inhibiting Atherosclerosis and Markers of Inflammation in Atherosclerotic Development in Apolipoprotein-E Knockout Mice.

    PubMed

    Li, Rong-Juan; Sun, Yan; Wang, Qin; Yang, Jiao; Yang, Ya; Song, Li; Wang, Zheng; Luo, Xiang-Hong; Su, Rui-Juan

    2015-08-01

    We sought to validate the hypothesis that the development of atherosclerosis can be suppressed by the interleukin-1 receptor antagonist (IL-1Ra) in murine models of atherosclerosis in vivo, noninvasively seen by means of high-resolution ultrasound biomicroscopy, and we studied changes in inflammatory markers such as IL-1 and C-reactive protein (CRP) plasma levels in these models of atherosclerosis. We divided IL-1Ra(+/-)/apolipoprotein-E (apoE)(-/-) and IL-1Ra(+/+)/apoE(-/-) mice into 2 age groups, used as atherosclerotic models. The control groups were age-matched IL-1Ra(+/+)/apoE(+/+) mice. Plaque thickness was measured in the ascending aorta in short-axis images by means of ultrasound and histology. Plasma levels of IL-1 and CRP were quantified in the 3 murine groups. At 16 weeks, plaque thickness in the ascending aortas of the IL-1Ra(+/-)/apoE(-/-) mice was significantly greater than that in the IL-1Ra(+/+)/apoE(-/-) mice, on ultrasound and histology (P <0.01). In contrast, at 32 weeks, the differences between these 2 genotypes were not statistically significant. Serum IL-1 levels were lower in the IL-1Ra(+/-)/apoE(-/-) mice than in the IL-1Ra(+/+)/apoE(-/-) mice at 16 and 32 weeks (P <0.05). At 16 weeks, serum CRP levels in the IL-1Ra(+/-)/apoE(-/-) mice were higher than in the IL-1Ra(+/+)/apoE(-/-) mice (P <0.01). Our results suggest that ultrasound biomicroscopy enables evaluation of atherosclerotic lesions in vivo, noninvasively and in real-time, in apoE(-/-) mice. Partial IL-1Ra deficiencies might promote early plaque development in 16-week-old apoE(-/-) mice. The balance of IL-1 and IL-1Ra might influence atherosclerotic development. Finally, CRP might affect the initiation of atherosclerosis, rather than its progression. PMID:26413013

  11. Somatostatin receptor 2 knockout/lacZ knockin mice show impaired motor coordination and reveal sites of somatostatin action within the striatum.

    PubMed

    Allen, Jeremy P; Hathway, Gareth J; Clarke, Neil J; Jowett, Mike I; Topps, Stephanie; Kendrick, Keith M; Humphrey, Patrick P A; Wilkinson, Lawrence S; Emson, Piers C

    2003-05-01

    The peptide somatostatin can modulate the functional output of the basal ganglia. The exact sites and mechanisms of this action, however, are poorly understood, and the physiological context in which somatostatin acts is unknown. Somatostatin acts as a neuromodulator via a family of five 7-transmembrane G protein-coupled receptors, SSTR1-5, one of which, SSTR2, is known to be functional in the striatum. We have investigated the role of SSTR2 in basal ganglia function using mice in which Sstr2 has been inactivated and replaced by the lacZ reporter gene. Analysis of Sstr2lacZ expression in the brain by beta-galactosidase histochemistry demonstrated a widespread pattern of expression. By comparison to previously published in situ hybridization and immunohistochemical data, Sstr2lacZ expression was shown to accurately recapitulate that of Sstr2 and thus provided a highly sensitive model to investigate cell-type-specific expression of Sstr2. In the striatum, Sstr2 expression was identified in medium spiny projection neurons restricted to the matrix compartment and in cholinergic interneurons. Sstr2 expression was not detected in any other nuclei of the basal ganglia except for a sparse number of nondopaminergic neurons in the substantia nigra. Microdialysis in the striatum showed Sstr2-null mice were selectively refractory to somatostatin-induced dopamine and glutamate release. In behavioural tests, Sstr2-null mice showed normal levels of locomotor activity and normal coordination in undemanding tasks. However, in beam-walking, a test of fine motor control, Sstr2-null mice were severely impaired. Together these data implicate an important neuromodulatory role for SSTR2 in the striatum. PMID:12752788

  12. Effects of chemokine receptor signalling on cognition-like, emotion-like and sociability behaviours of CCR6 and CCR7 knockout mice.

    PubMed

    Jaehne, E J; Baune, B T

    2014-03-15

    Inflammation is regarded as an important mechanism of neuropsychiatric disorders. Chemokines, which are a part of the immune system, have effects on various aspects of brain function, but little is known about their effects on behaviour. We have compared the cognition-like behaviour (learning and spatial memory) of CCR6(-/-) and CCR7(-/-) mice with wild type (WT) C57BL/6 mice, in the Barnes maze, as well as a range of other behaviours, including exploratory, anxiety and depression-like behaviour, using a battery of tests. Levels of cytokines TNF-α, IL-1β and IL-6 were also measured. In the Barnes maze, CCR7(-/-) mice were shown to take longer to learn the location of the escape box on the 1st of 4 days of training. In the behavioural battery, CCR6(-/-) mice showed higher locomotor activity and lower anxiety in the open field test, and a lack of preference for social novelty in a sociability test. CCR7(-/-) mice behaved much like WT mice, although showed higher anxiety in Elevated Zero Maze. While baseline saccharin preference in a 2-bottle choice test, a test for anhedonia depression-like behaviour, was equal in all strains at baseline, weekly tests showed that both CCR6(-/-) and CCR7(-/-) mice developed a decreased preference for saccharin compared to WT over time. There were no differences between strains in any of the cytokines measured. These results suggest that chemokine receptors may play a role in cognition and learning behaviour, as well as anxiety and other behaviours, although the biological mechanisms are still unclear.

  13. Performance Deficits of NK1 Receptor Knockout Mice in the 5-Choice Serial Reaction-Time Task: Effects of d-Amphetamine, Stress and Time of Day

    PubMed Central

    Yan, Ting Carrie; Dudley, Julia A.; Weir, Ruth K.; Grabowska, Ewelina M.; Peña-Oliver, Yolanda; Ripley, Tamzin L.; Hunt, Stephen P.; Stephens, David N.; Stanford, S. Clare

    2011-01-01

    Background The neurochemical status and hyperactivity of mice lacking functional substance P-preferring NK1 receptors (NK1R-/-) resemble abnormalities in Attention Deficit Hyperactivity Disorder (ADHD). Here we tested whether NK1R-/- mice express other core features of ADHD (impulsivity and inattentiveness) and, if so, whether they are diminished by d-amphetamine, as in ADHD. Prompted by evidence that circadian rhythms are disrupted in ADHD, we also compared the performance of mice that were trained and tested in the morning or afternoon. Methods and Results The 5-Choice Serial Reaction-Time Task (5-CSRTT) was used to evaluate the cognitive performance of NK1R-/- mice and their wildtypes. After training, animals were tested using a long (LITI) and a variable (VITI) inter-trial interval: these tests were carried out with, and without, d-amphetamine pretreatment (0.3 or 1 mg/kg i.p.). NK1R-/- mice expressed greater omissions (inattentiveness), perseveration and premature responses (impulsivity) in the 5-CSRTT. In NK1R-/- mice, perseveration in the LITI was increased by injection-stress but reduced by d-amphetamine. Omissions by NK1R-/- mice in the VITI were unaffected by d-amphetamine, but premature responses were exacerbated by this psychostimulant. Omissions in the VITI were higher, overall, in the morning than the afternoon but, in the LITI, premature responses of NK1R-/- mice were higher in the afternoon than the morning. Conclusion In addition to locomotor hyperactivity, NK1R-/- mice express inattentiveness, perseveration and impulsivity in the 5-CSRTT, thereby matching core criteria for a model of ADHD. Because d-amphetamine reduced perseveration in NK1R-/- mice, this action does not require functional NK1R. However, the lack of any improvement of omissions and premature responses in NK1R-/- mice given d-amphetamine suggests that beneficial effects of this psychostimulant in other rodent models, and ADHD patients, need functional NK1R. Finally, our results

  14. Leptin receptor-deficient (knockout) medaka, Oryzias latipes, show chronical up-regulated levels of orexigenic neuropeptides, elevated food intake and stage specific effects on growth and fat allocation.

    PubMed

    Chisada, Shin-ichi; Kurokawa, Tadahide; Murashita, Koji; Rønnestad, Ivar; Taniguchi, Yoshihito; Toyoda, Atsushi; Sakaki, Yoshiyuki; Takeda, Shunichi; Yoshiura, Yasutoshi

    2014-01-01

    The first studies that identified leptin and its receptor (LepR) in mammals were based on mutant animals that displayed dramatic changes in body-weight and regulation of energy homeostasis. Subsequent studies have shown that a deficiency of leptin or LepR in homoeothermic mammals results in hyperphagia, obesity, infertility and a number of other abnormalities. The physiological roles of leptin-mediated signaling in ectothermic teleosts are still being explored. Here, we produced medaka with homozygous LepR gene mutation using the targeting induced local lesions in a genome method. This knockout mutant had a point mutation of cysteine for stop codon at the 357th amino acid just before the leptin-binding domain. The evidence for loss of function of leptin-mediated signaling in the mutant is based on a lack of response to feeding in the expression of key appetite-related neuropeptides in the diencephalon. The mutant lepr−/− medaka expressed constant up-regulated levels of mRNA for the orexigenic neuropeptide Ya and agouti-related protein and a suppressed level of anorexigenic proopiomelanocortin 1 in the diencephalon independent of feeding, which suggests that the mutant did not possess functional LepR. Phenotypes of the LepR-mutant medaka were analyzed in order to understand the effects on food intake, growth, and fat accumulation in the tissues. The food intake of the mutant medaka was higher in post-juveniles and adult stages than that of wild-type (WT) fish. The hyperphagia led to a high growth rate at the post-juvenile stage, but did not to significant alterations in final adult body size. There was no additional deposition of fat in the liver and muscle in the post-juvenile and adult mutants, or in the blood plasma in the adult mutant. However, adult LepR mutants possessed large deposits of visceral fat, unlike in the WT fish, in which there were none. Our analysis confirms that LepR in medaka exert a powerful influence on the control on food intake. Further

  15. Knockout beyond the dripline

    SciTech Connect

    Bonaccorso, A.; Charity, R. J.; Kumar, R.; Salvioni, G.

    2015-02-24

    In this contribution, we will describe neutron and proton removal from {sup 9}C and {sup 7}Be which are two particularly interesting nuclei entering the nucleo-synthesis pp-chain [1, 2]. Neutron and proton removal reactions have been used in the past twenty years to probe the single-particle structure of exotic nuclei. The core parallel-momentum distribution can give information on the angular momentum and spin of the nucleon initial state while the total removal cross section is sensitive to the asymptotic part of the initial wave function and also to the reaction mechanism. Because knockout is a peripheral reaction from which the Asymptotic Normalization Constant (ANC) of the single-particle wave function can be extracted, it has been used as an indirect method to obtain the rate of reactions like {sup 8}B(p,γ){sup 9}C or {sup 7}Be(p,γ){sup 8}B. Nucleon removal has recently been applied by the HiRA collaboration [3] to situations in which the remaining “core” is beyond the drip line, such as {sup 8}C and {sup 6}Be, unbound by one or more protons, and whose excitation-energy spectrum can be obtained by the invariant-mass method. By gating on the ground-state peak, “core” parallel-momentum distributions and total knockout cross sections have been obtained similar to previous studies with well-bound “cores”. In addition for each projectile, knock out to final bound states has also been obtained in several cases. We will report on the theoretical description and comparison to this experimental data for a few cases for which advances in the accuracy of the transfer-to-the continuum model [4, 5] have been made [6]. These include the use, when available, of “ab-initio” overlaps for the initial state [7] and in particular their ANC values [8]. Also, the construction of a nucleus-target folding potential for the treatment of the core-target S-matrix [9] using for the cores “ab-initio” densities [10] and state-of-the-art n−{sup 9}Be optical

  16. 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.

  17. Effect of homologous serotonin receptor loop substitutions on the heterologous expression in Pichia of a chimeric acetylcholine-binding protein with alpha-bungarotoxin-binding activity.

    PubMed

    Paulo, Joao A; Hawrot, Edward

    2009-10-01

    The molluscan acetylcholine-binding protein (AChBP) is a soluble homopentameric homolog of the extracellular domain of various ligand-gated ion channels. Previous studies have reported that AChBP, when fused to the ion pore domain of the serotonin receptor (5HT(3A)R), can form a functional ligand-gated chimeric channel only if the AChBP loop regions between beta-strands beta1 and beta2 (beta1-beta2), beta6 and beta7 (beta6-beta7), and beta8 and beta9 (beta8-beta9) are replaced with those of the 5HT(3A)R. To investigate further the potential interactions among these three important loop regions in a membrane- and detergent-free system, we designed AChBP constructs in which loops beta1-beta2, beta6-beta7, and beta8-beta9 of the AChBP were individually and combinatorially substituted in all permutations with the analogous loops of the 5HT(3A)R. These chimeras were expressed as secreted proteins using the Pichia pastoris yeast expression system. [(125)I]-alpha-Bungarotoxin-binding was detected in the culture media obtained from homologous recombinant clones expressing the wild-type AChBP, the beta1-beta2 loop-only chimera, and the chimera containing all three 5HT(3A)R loop substitutions. The remaining chimeras failed to show [(125)I]-alpha-bungarotoxin binding, and further analysis of cellular extracts allowed us to determine that these binding-negative chimeric constructs accumulated intracellularly and were not secreted into the culture medium. Our results demonstrate that coordinated interactions among loops beta1-beta2, beta6-beta7, and beta8-beta9 are essential for the formation of a functional ligand-binding site, as evidenced by [(125)I]-alpha-bungarotoxin-binding, and for efficient protein secretion. In addition, the constructs described here demonstrate the feasibility of utilizing soluble scaffolds to explore functionally important interactions within the extracellular domain of membrane-bound proteins. PMID:19427904

  18. Altered dendritic distribution of dopamine D2 receptors and reduction in mitochondrial number in parvalbumin-containing interneurons in the medial prefrontal cortex of cannabinoid-1 (CB1) receptor knockout mice

    PubMed Central

    Fitzgerald, Megan L.; Chan, June; Mackie, Kenneth; Lupica, Carl R.; Pickel, Virginia M.

    2013-01-01

    The prelimbic prefrontal cortex (PL) is a brain region integral to complex behaviors that are highly influenced by cannabinoids and by dopamine D2 receptor (D2R)-mediated regulation of fast-firing parvalbumin-containing interneurons. We have recently shown that constitutive deletion of the cannabinoid CB1 receptor (CB1R) greatly reduces parvalbumin levels in these neurons. The effects of CB1R deletion on PL parvalbumin interneurons may be ascribed to loss of CB1R-mediated retrograde signaling on mesocortical dopamine transmission, and, in turn, altered expression and/or subcellular distribution of the D2R in the PL. Furthermore, diminished parvalbumin expression could indicate metabolic changes in fast-firing interneurons that may be reflected in changes in mitochondrial density in this population. We therefore comparatively examined electron microscopic dual labeling of the D2R and parvalbumin in CB1 (−/−) and CB1 (+/+) mice to test the hypothesis that absence of the CB1R produces changes in D2R localization and mitochondrial distribution in parvalbumin-containing interneurons of the PL. CB1 (−/−) mice had a significantly lower density of cytoplasmic D2R-immunogold particles in medium parvalbumin-labeled dendrites and a concomitant increase in the density of these particles in small dendrites. These dendrites received both excitatory and inhibitory-type synapses from unlabeled terminals and contained many mitochondria, whose numbers were significantly reduced in the CB1 (−/−) mice. Non-parvalbumin containing dendrites showed no between-group differences in either D2R distribution or mitochondrial number. These results suggest that cannabinoid signaling provides an important determinant of dendritic D2 receptor distribution and mitochondrial availability in fast-spiking interneurons. PMID:22592925

  19. In vivo interaction of steroid receptor coactivator (SRC)-1 and the activation function-2 domain of the thyroid hormone receptor (TR) beta in TRbeta E457A knock-in and SRC-1 knockout mice.

    PubMed

    Alonso, Manuela; Goodwin, Charles; Liao, Xiaohui; Ortiga-Carvalho, Tania; Machado, Danielle S; Wondisford, Fredric E; Refetoff, Samuel; Weiss, Roy E

    2009-08-01

    The activation function-2 (AF-2) domain of the thyroid hormone (TH) receptor (TR)-beta is a TH-dependent binding site for nuclear coactivators (NCoA), which modulate TH-dependent gene transcription. In contrast, the putative AF-1 domain is a TH-independent region interacting with NCoA. We determined the specificity of the AF-2 domain and NCoA interaction by evaluating thyroid function in mice with combined disruption of the AF-2 domain in TRbeta, due to a point mutation (E457A), and deletion of one of the NCoAs, steroid receptor coactivator (SRC)-1. The E457A mutation was chosen because it abolishes NCoA recruitment in vitro while preserving normal TH binding and corepressor interactions resulting in resistance to TH. At baseline, disruption of SRC-1 in the homozygous knock-in (TRbeta(E457A/E457A)) mice worsened the degree of resistance to TH, resulting in increased serum T(4) and TSH. During TH deprivation, disruption of AF-2 and SRC-1 resulted in a TSH rise 50% of what was seen when AF-2 alone was removed, suggesting that SRC-1 was interacting outside of the AF-2 domain. Therefore, 1) during TH deprivation, SRC-1 is necessary for activating the hypothalamic-pituitary-thyroid axis; 2) ligand-dependent repression of TSH requires an intact AF-2; and 3) SRC-1 may interact with the another region of the TRbeta or the TRalpha to regulate TH action in the pituitary. This report demonstrates the dual interaction of NCoA in vivo: the TH-independent up-regulation possibly through another domain and TH-dependent down-regulation through the AF-2 domain. PMID:19406944

  20. Central nervous system-specific knockout of steroidogenic factor 1.

    PubMed

    Kim, Ki Woo; Zhao, Liping; Parker, Keith L

    2009-03-01

    Steroidogenic factor 1 (SF-1) is a nuclear receptor that plays important roles in the hypothalamus-pituitary-steroidogenic organ axis. Global knockout studies in mice revealed the essential in vivo roles of SF-1 in the ventromedial hypothalamic (VMH) nucleus, adrenal glands, and gonads. One limitation of global SF-1 knockout mice is their early postnatal death from adrenocortical insufficiency. To overcome limitations of the global knockout mice and to delineate the roles of SF-1 in the brain, we used Cre/loxP recombination technology to genetically ablate SF-1 specifically in the central nervous system (CNS). Mice with CNS-specific knockout of SF-1 mediated by nestin-Cre showed increased anxiety-like behavior, revealing a crucial role of SF-1 in a complex behavioral phenotype. Our studies with CNS-specific SF-1 KO mice also defined roles of SF-1 in regulating the VMH expression of target genes implicated in anxiety and energy homeostasis. Therefore, this review will focus on our recent studies defining the functional roles of SF-1 in the VMH linked to anxiety and energy homeostasis.

  1. Expression of serotonin receptor genes in cranial ganglia.

    PubMed

    Maeda, Naohiro; Ohmoto, Makoto; Yamamoto, Kurumi; Kurokawa, Azusa; Narukawa, Masataka; Ishimaru, Yoshiro; Misaka, Takumi; Matsumoto, Ichiro; Abe, Keiko

    2016-03-23

    Taste cells release neurotransmitters to gustatory neurons to transmit chemical information they received. Sweet, umami, and bitter taste cells use ATP as a neurotransmitter. However, ATP release from sour taste cells has not been observed so far. Instead, they release serotonin when they are activated by sour/acid stimuli. Thus it is still controversial whether sour taste cells use ATP, serotonin, or both. By reverse transcription-polymerase chain reaction and subsequent in situ hybridization (ISH) analyses, we revealed that of 14 serotonin receptor genes only 5-HT3A and 5-HT3B showed significant/clear signals in a subset of neurons of cranial sensory ganglia in which gustatory neurons reside. Double-fluorescent labeling analyses of ISH for serotonin receptor genes with wheat germ agglutinin (WGA) in cranial sensory ganglia of pkd1l3-WGA mice whose sour neural pathway is visualized by the distribution of WGA originating from sour taste cells in the posterior region of the tongue revealed that WGA-positive cranial sensory neurons rarely express either of serotonin receptor gene. These results suggest that serotonin receptors expressed in cranial sensory neurons do not play any role as neurotransmitter receptor from sour taste cells. PMID:26854841

  2. 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

  3. [Genetic knockout--the first steps and outlook for a neurophysiology of behavior].

    PubMed

    Popova, N K

    2000-01-01

    A review of modern data on genetic knockout strategy application to study the brain neurotransmitters and their role in the regulation of behavior. Advantages and shortcomings of genetic knockout of receptors and the enzymes of neurotransmitters metabolism models in comparison to other methods are discussed. Data on the effect of genetic knockout of various types of opioid, dopamine, serotonin and adrenoreceptors as well as enzymes in biosynthesis of catecholamines and serotonin on physiology and behavior is adduced. The data provide evidence that genetic knockout reproduces a principal effects of the lack of receptors and enzymes and allows to find new yet unknown properties. Mouse strains with genetic knockout represent unique models of hereditary neuropathology. At the same time the data presented clearly demonstrated that the lack of a single type of receptor or enzyme does not lead usually to disorganization of regulated by them physiological functions and behavior. The data witness to the complexity and multifactoriality of their regulations and evidenced the great compensatory potentials of organism.

  4. Enhanced Long-Term and Impaired Short-Term Spatial Memory in GluA1 AMPA Receptor Subunit Knockout Mice: Evidence for a Dual-Process Memory Model

    ERIC Educational Resources Information Center

    Sanderson, David J.; Good, Mark A.; Skelton, Kathryn; Sprengel, Rolf; Seeburg, Peter H.; Rawlins, J. Nicholas P.; Bannerman, David M.

    2009-01-01

    The GluA1 AMPA receptor subunit is a key mediator of hippocampal synaptic plasticity and is especially important for a rapidly-induced, short-lasting form of potentiation. GluA1 gene deletion impairs hippocampus-dependent, spatial working memory, but spares hippocampus-dependent spatial reference memory. These findings may reflect the necessity of…

  5. 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.

  6. Pathway knockout and redundancy in metabolic networks.

    PubMed

    Min, Yong; Jin, Xiaogang; Chen, Ming; Pan, Zhengzheng; Ge, Ying; Chang, Jie

    2011-02-01

    The robustness and stability of complex cellular networks is often attributed to the redundancy of components, including genes, enzymes and pathways. Estimation of redundancy is still an open question in systems biology. Current theoretical tools to measure redundancy have various strengths and shortcomings in providing a comprehensive description of metabolic networks. Specially, there is a lack of effective measures to cover different perturbation situations. Here we present a pathway knockout algorithm to improve quantitative measure of redundancy in metabolic networks grounded on the elementary flux mode (EFM) analysis. The proposed redundancy measure is based on the average ratio of remaining EFMs after knockout of one EFM in the unperturbed state. We demonstrated with four example systems that our algorithm overcomes limits of previous measures, and provides additional information about redundancy in the situation of targeted attacks. Additionally, we compare existing enzyme knockout and our pathway knockout algorithm by the mean-field analysis, which provides mathematical expression for the average ratio of remaining EFMs after both types of knockout. Our results prove that multiple-enzymes knockout does not always yield more information than single-enzyme knockout for evaluating redundancy. Indeed, pathway knockout considers additional effects of structural asymmetry. In the metabolic networks of amino acid anabolism in Escherichia coli and human hepatocytes, and the central metabolism in human erythrocytes, we validate our mean-field solutions and prove the capacity of pathway knockout algorithm. Moreover, in the E. coli model the two sub-networks synthesizing amino acids that are essential and those that are non-essential for humans are studied separately. In contrast to previous studies, we find that redundancy of two sub-networks is similar with each other, and even sub-networks synthesizing essential amino acids can be more redundant.

  7. Enhanced cognitive performance of dopamine D3 receptor "knock-out" mice in the step-through passive-avoidance test: assessing the role of the endocannabinoid/endovanilloid systems.

    PubMed

    Micale, Vincenzo; Cristino, Luigia; Tamburella, Alessandra; Petrosino, Stefania; Leggio, Gian Marco; Di Marzo, Vincenzo; Drago, Filippo

    2010-06-01

    Increasing evidence suggests a pivotal role of the D3 receptor (D3R) in cognitive processes and the involvement of endocannabinoid/endovanilloid signaling in the pathophysiology of neurodegenerative disorders such as Alzheimer's disease. This study was undertaken to investigate both the basal and beta-amyloid peptide 1-42 (BAP 1-42)-impaired cognitive performance of D3R((-/-)) mice, and the role therein of endocannabinoids/endovanilloids. D3R((-/-)) mice were either untreated or injected i.c.v. with 400 pMol BAP 1-42 or vehicle to be tested 14 days later in a step-through passive-avoidance paradigm. The CB(1) receptor antagonist, rimonabant (1mg/kg), or the transient receptor potential vanilloid-type 1 channel (TRPV1) antagonist SB366791, were injected intraperitoneally for 11 or 7 days. The retention test was performed 1, 7 and 14 days after the learning trial. Wild-type (WT) mice were subjected to the same procedures. D3R((-/-)) mice exhibited a better basal cognitive performance as compared to WT mice (p<0.001), which was reversed by TRPV1 antagonism. BAP 1-42 induced a pronounced worsening of the passive-avoidance response in all tests and in both genotypes (p<0.001). Rimonabant treatment never affected the cognitive performance of healthy mice, but fully counteracted BAP 1-42-induced amnesic effects in both D3R((-/-)) and WT mice only when administered for 11 days, whereas, when administered for 7 days, only transiently affected WT mice and caused more prolonged cognitive ameliorations in D3R((-/-)) mice. These results support the involvement of D3R and TRPV1 in cognitive processes and the concept that A beta peptides inhibit memory retention in mice through the involvement of endocannabinoids.

  8. Studying TGF-beta superfamily signaling by knockouts and knockins.

    PubMed

    Chang, H; Lau, A L; Matzuk, M M

    2001-06-30

    The transforming growth factor beta (TGF-beta) superfamily has profound effects on many aspects of animal development. In the last decade, our laboratory and others have performed in vivo functional studies on multiple components of the TGF-beta superfamily signal transduction pathway, including upstream ligands, transmembrane receptors, receptor-associated proteins and downstream Smad proteins. We have taken gene knockout approaches to generate null alleles of the genes of interest, as well as a gene knockin approach to replace the mature region of one TGF-beta superfamily ligand with another. We found that activin betaB, expressed in the spatiotemporal pattern of activin betaA, can function as a hypomorphic allele of activin betaA and rescue the craniofacial defects and neonatal lethal phenotype of activin betaA-deficient mice. With the knockout approach, we have shown that the expression pattern of a component in the TGF-beta superfamily signal transduction cascade does not necessarily predict its in vivo function. Two liver-specific activins, activin betaC and activin betaE are dispensable for liver development, regeneration and function, whereas ubiquitously expressed Smad5 has specific roles in the development of multiple embryonic and extraembryonic tissues. PMID:11451570

  9. 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.

  10. Microarray analysis of E9.5 reduced folate carrier (RFC1; Slc19a1) knockout embryos reveals altered expression of genes in the cubilin-megalin multiligand endocytic receptor complex

    PubMed Central

    Gelineau-van Waes, Janee; Maddox, Joyce R; Smith, Lynette M; van Waes, Michael; Wilberding, Justin; Eudy, James D; Bauer, Linda K; Finnell, Richard H

    2008-01-01

    Background The reduced folate carrier (RFC1) is an integral membrane protein and facilitative anion exchanger that mediates delivery of 5-methyltetrahydrofolate into mammalian cells. Adequate maternal-fetal transport of folate is necessary for normal embryogenesis. Targeted inactivation of the murine RFC1 gene results in post-implantation embryolethality, but daily folic acid supplementation of pregnant dams prolongs survival of homozygous embryos until mid-gestation. At E10.5 RFC1-/- embryos are developmentally delayed relative to wildtype littermates, have multiple malformations, including neural tube defects, and die due to failure of chorioallantoic fusion. The mesoderm is sparse and disorganized, and there is a marked absence of erythrocytes in yolk sac blood islands. The identification of alterations in gene expression and signaling pathways involved in the observed dysmorphology following inactivation of RFC1-mediated folate transport are the focus of this investigation. Results Affymetrix microarray analysis of the relative gene expression profiles in whole E9.5 RFC1-/- vs. RFC1+/+ embryos identified 200 known genes that were differentially expressed. Major ontology groups included transcription factors (13.04%), and genes involved in transport functions (ion, lipid, carbohydrate) (11.37%). Genes that code for receptors, ligands and interacting proteins in the cubilin-megalin multiligand endocytic receptor complex accounted for 9.36% of the total, followed closely by several genes involved in hematopoiesis (8.03%). The most highly significant gene network identified by Ingenuity™ Pathway analysis included 12 genes in the cubilin-megalin multiligand endocytic receptor complex. Altered expression of these genes was validated by quantitative RT-PCR, and immunohistochemical analysis demonstrated that megalin protein expression disappeared from the visceral yolk sac of RFC1-/- embryos, while cubilin protein was widely misexpressed. Conclusion Inactivation of

  11. Hazara virus infection is lethal for adult type I interferon receptor-knockout mice and may act as a surrogate for infection with the human-pathogenic Crimean-Congo hemorrhagic fever virus.

    PubMed

    Dowall, Stuart D; Findlay-Wilson, Stephen; Rayner, Emma; Pearson, Geoff; Pickersgill, Janice; Rule, Antony; Merredew, Natasha; Smith, Hazel; Chamberlain, John; Hewson, Roger

    2012-03-01

    Hazara virus (HAZV) is closely related to the Crimean-Congo hemorrhagic fever virus (CCHFV). HAZV has not been reported to cause human disease; work with infectious material can be carried out at containment level (CL)-2. By contrast, CCHFV causes a haemorrhagic fever in humans and requires CL-4 facilities. A disease model of HAZV infection in mice deficient in the type I interferon receptor is reported in this study. Dose-response effects were seen with higher doses, resulting in a shorter time to death and earlier detection of viral loads in organs. The lowest dose of 10 p.f.u. was still lethal in over 50 % of the mice. Histopathological findings were identified in the liver, spleen and lymph nodes, with changes similar to a recent mouse model of CCHFV infection. The findings demonstrate that inoculation of mice with HAZV may act as a useful surrogate model for the testing of antiviral agents against CCHFV.

  12. The angiotensin converting enzyme inhibitor, captopril, prevents the hyperactivity and impulsivity of neurokinin-1 receptor gene 'knockout' mice: sex differences and implications for the treatment of attention deficit hyperactivity disorder.

    PubMed

    Porter, Ashley J; Pillidge, Katharine; Grabowska, Ewelina M; Stanford, S Clare

    2015-04-01

    Mice lacking functional neurokinin-1 receptors (NK1R-/-) display behavioural abnormalities resembling attention deficit hyperactivity disorder (ADHD): locomotor hyperactivity, impulsivity and inattentiveness. The preferred ligand for NK1R, substance P, is metabolised by angiotensin converting enzyme (ACE), which forms part of the brain renin angiotensin system (BRAS). In view of evidence that the BRAS modulates locomotor activity and cognitive performance, we tested the effects of drugs that target the BRAS on these behaviours in NK1R-/- and wildtype mice. We first tested the effects of the ACE inhibitor, captopril, on locomotor activity. Because there are well-established sex differences in both ADHD and ACE activity, we compared the effects of captopril in both male and female mice. Locomotor hyperactivity was evident in male NK1R-/- mice, only, and this was abolished by treatment with captopril. By contrast, male wildtypes and females of both genotypes were unaffected by ACE inhibition. We then investigated the effects of angiotensin AT1 (losartan) and AT2 (PD 123319) receptor antagonists on the locomotor activity of male NK1R-/- and wildtype mice. Both antagonists increased the locomotor activity of NK1R-/- mice, but neither affected the wildtypes. Finally, we tested the effects of captopril on the performance of male NK1R-/- and wildtype mice in the 5-choice serial reaction-time task (5-CSRTT) and found that ACE inhibition prevented the impulsivity of NK1R-/- mice. These results indicate that certain behaviours, disrupted in ADHD, are influenced by an interaction between the BRAS and NK1R, and suggest that ACE inhibitors could provide a novel treatment for this disorder.

  13. 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).

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. Gonadotropin Signaling in Zebrafish Ovary and Testis Development: Insights From Gene Knockout Study.

    PubMed

    Chu, Lianhe; Li, Jianzhen; Liu, Yun; Cheng, Christopher H K

    2015-12-01

    Using the transcription activator-like effectors nucleases-mediated gene knockout technology, we have previously demonstrated that LH signaling is required for oocyte maturation and ovulation but is dispensable for testis development in zebrafish. Here, we have further established the fshb and fshr knockout zebrafish lines. In females, fshb mutant is subfertile, whereas fshr mutant is infertile. Folliculogenesis is partially affected in the fshb mutant but is completely arrested at the primary growth stage in the fshr mutant. In males, fshb and fshr mutant are fertile. The fertilization rate and histological structure of the testis is not affected. However, double knockout of fshb;lhb or fshr;lhr leads to all infertile male offspring. The key steroid hormones and steroidogenic genes are dramatically decreased in double knockout mutant (fshb;lhb and fshr;lhr) but not in single knockout mutant (fshb, lhb, fshr, and lhr) males. Furthermore, we have also demonstrated the constitutive activities of both FSH receptor (FSHR) and LH receptor in zebrafish and the compensatory role of LH by cross-reacting with FSHR in the fshb;lhr double mutant, thus explaining the phenotypic discrepancy observed among the ligand/receptor mutant lines. Taken together, our data established the following models on the roles of gonadotropin signaling in zebrafish gonad development. In females, FSH signaling is mainly responsible for promoting follicular growth, whereas LH signaling is mainly responsible for stimulating oocyte maturation and ovulation. In males, the functions of FSH and LH signaling overlap, and only disruption of both FSH and LH signaling could lead to the infertile phenotype. In the absence of FSH, LH could play a compensatory role by cross-reacting with FSHR in both male and female.

  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. Nuclear receptors constitutive androstane receptor and pregnane X receptor ameliorate cholestatic liver injury

    PubMed Central

    Stedman, Catherine A. M.; Liddle, Christopher; Coulter, Sally A.; Sonoda, Junichiro; Alvarez, Jacqueline G. A.; Moore, David D.; Evans, Ronald M.; Downes, Michael

    2005-01-01

    Cholestasis is associated with accumulation of bile acids and lipids, and liver injury. The constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are xenobiotic nuclear receptors that coordinate protective hepatic responses to potentially toxic stimuli, including bile acids. We investigated the role of these receptors in the regulation of bile acid and lipid metabolism in a bile duct ligation (BDL) model of cholestasis applied to receptor knockout mice. Hepatic damage from bile acid accumulation was increased in both CAR knockout (CARKO) and PXR knockout mice, but bile acid concentrations were lower in CARKO mice. High-density lipoprotein (HDL) cholesterol was elevated in CARKO mice, and serum total cholesterol increased less in CARKO or PXR knockout mice than WT mice after BDL. Gene expression analysis of the BDL knockout animals demonstrated that, in response to cholestasis, PXR and CAR both repressed and induced the specific hepatic membrane transporters Oatp-c (organic anion transporting polypeptide C) and Oatp2 (Na+-dependent organic anion transporter 2), respectively. Induction of the xenobiotic transporter multidrug resistance protein 1 in cholestasis was independent of either PXR or CAR, in contrast to the known pattern of induction of multidrug resistance protein 1 by xenobiotics. These results demonstrate that CAR and PXR influence cholesterol metabolism and bile acid synthesis, as well as multiple detoxification pathways, and suggest their potential role as therapeutic targets for the treatment of cholestasis and lipid disorders. PMID:15684063

  1. Cloning and expression of ligand-gated ion-channel receptor L2 in central nervous system

    SciTech Connect

    Houtani, Takeshi; Munemoto, Yumi; Kase, Masahiko; Sakuma, Satoru; Tsutsumi, Toshiyuki; Sugimoto, Tetsuo . E-mail: sugimoto@takii.kmu.ac.jp

    2005-09-23

    An orphan receptor of ligand-gated ion-channel type (L2, also termed ZAC according to the presence of zinc ion for channel activation) was identified by computer-assisted search programs on human genome database. The L2 protein shares partial homology with serotonin receptors 5HT3A and 5HT3B. We have cloned L2 cDNA derived from human caudate nucleus and characterized the exon-intron structure as follows: (1) The L2 protein has four transmembrane regions (M1-M4) and a long cytoplasmic loop between M3 and M4. (2) The sequence is conserved in species including chimpanzee, dog, cow, and opossum. (3) Nine exons form its protein-coding region and especially exon 5 corresponds to a disulfide bond region on the amino-terminal side. Our analysis using multiple tissue cDNA panels revealed that at least two splicing variants of L2 mRNA are present. The cDNA PCR amplification study revealed that L2 mRNA is expressed in tissues including brain, pancreas, liver, lung, heart, kidney, and skeletal muscle while 5HT3A mRNA could be detected in brain, heart, placenta, lung, kidney, pancreas, and skeletal muscle, and 5HT3B mRNA in brain, kidney, and skeletal muscle, suggesting different significance in tissue expression of these receptors. Regional expression of L2 mRNA and protein was examined in brain. The RT-PCR studies confirmed L2 mRNA expression in hippocampus, striatum, amygdala, and thalamus in adult brain. The L2 protein was immunolocalized by using antipeptide antibodies. Immunostained tissue sections revealed that L2-like immunoreactivity was dominantly expressed in the hippocampal CA3 pyramidal cells and in the polymorphic layer of the dentate gyrus. We analyzed the expression of L2 protein in HEK293 cells using GFP fusion protein reporter system. Western blots revealed that L2 protein confers sugar chains on the extracellular side. In transfected HEK293 cells, cellular membranes and intracellular puncta were densely labeled with GFP, suggesting selective dispatch to the

  2. N-Benzylpiperidine Derivatives as α7 Nicotinic Receptor Antagonists.

    PubMed

    Criado, Manuel; Mulet, José; Sala, Francisco; Sala, Salvador; Colmena, Inés; Gandía, Luis; Bautista-Aguilera, Oscar M; Samadi, Abdelouahid; Chioua, Mourad; Marco-Contelles, José

    2016-08-17

    A series of multitarget directed propargylamines, as well as other differently susbstituted piperidines have been screened as potential modulators of neuronal nicotinic acetylcholine receptors (nAChRs). Most of them showed antagonist actions on α7 nAChRs. Especially, compounds 13, 26, and 38 displayed submicromolar IC50 values on homomeric α7 nAChRs, whereas they were less effective on heteromeric α3β4 and α4β2 nAChRs (up to 20-fold higher IC50 values in the case of 13). Antagonism was concentration dependent and noncompetitive, suggesting that these compounds behave as negative allosteric modulators of nAChRs. Upon the study of a series of less complex derivatives, the N-benzylpiperidine motif, common to these compounds, was found to be the main pharmacophoric group. Thus, 2-(1-benzylpiperidin-4-yl)-ethylamine (48) showed an inhibitory potency comparable to the one of the previous compounds and also a clear preference for α7 nAChRs. In a neuroblastoma cell line, representative compounds 13 and 48 also inhibited, in a concentration-dependent manner, cytosolic Ca(2+) signals mediated by nAChRs. Finally, compounds 38 and 13 inhibited 5-HT3A serotonin receptors whereas they had no effect on α1 glycine receptors. Given the multifactorial nature of many pathologies in which nAChRs are involved, these piperidine antagonists could have a therapeutic potential in cases where cholinergic activity has to be negatively modulated. PMID:27254782

  3. 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

  4. 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.

  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. Mutations of L293 in transmembrane two of the mouse 5-hydroxytryptamine3A receptor alter gating and alcohol modulatory actions.

    PubMed

    Hu, Xiang-Qun; Hayrapetyan, Volodya; Gadhiya, Jay J; Rhubottom, Heather E; Lovinger, David M; Machu, Tina K

    2006-05-01

    1 The goal of this study was to determine whether mutations of L293 at the 15' position of TM2 in the 5-HT(3A) receptor alter macroscopic current kinetics, and if these changes could account for alterations in alcohol modulation. Receptor function was assessed in Xenopus oocytes under voltage-clamp and in HEK293 cells with whole-cell patch-clamp recording and rapid drug application. 2 Examination of responses of L293C and L293S receptors to agonist alone revealed enhanced activation, deactivation, and desensitization rates relative to the wild-type receptor. The L293G mutation produced marked slowing of deactivation and desensitization rates. Increased potency of 5-HT and increased efficacy of the partial agonist, DA, was also observed in these mutant receptors. 3 Ethanol and trichloroethanol (TCEt) enhancement of receptor function was reduced or eliminated in receptors containing L293 mutations to C, G, or S. The L293I mutant receptor retained ethanol and TCEt sensitivity. Ethanol and TCEt enhanced activation rate in the wild-type, but not the L293G and L293S receptors. No relationship was observed between any physicochemical property of the substituted amino acids and the change in alcohol potentiation of function. 4 The changes in receptor-channel properties in the mutant receptors support the idea that the L293 residue has important roles in channel gating. Our findings indicate that loss of allosteric modulation by alcohols is not related in any simple way to changes in channel kinetic properties brought about by L293 mutants. We did not observe any evidence that L293 is part of an alcohol binding site. PMID:16520747

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. K Basins floor sludge retrieval system knockout pot basket fuel burn accident

    SciTech Connect

    HUNT, J.W.

    1998-11-11

    The K Basins Sludge Retrieval System Preliminary Hazard Analysis Report (HNF-2676) identified and categorized a series of potential accidents associated with K Basins Sludge Retrieval System design and operation. The fuel burn accident was of concern with respect to the potential release of contamination resulting from a runaway chemical reaction of the uranium fuel in a knockout pot basket suspended in the air. The unmitigated radiological dose to an offsite receptor from this fuel burn accident is calculated to be much less than the offsite risk evaluation guidelines for anticipated events. However, because of potential radiation exposure to the facility worker, this accident is precluded with a safety significant lifting device that will prevent the monorail hoist from lifting the knockout pot basket out of the K Basin water pool.

  12. 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

  13. Universal statistics of the knockout tournament

    NASA Astrophysics Data System (ADS)

    Baek, Seung Ki; Yi, Il Gu; Park, Hye Jin; Kim, Beom Jun

    2013-11-01

    We study statistics of the knockout tournament, where only the winner of a fixture progresses to the next. We assign a real number called competitiveness to each contestant and find that the resulting distribution of prize money follows a power law with an exponent close to unity if the competitiveness is a stable quantity and a decisive factor to win a match. Otherwise, the distribution is found narrow. The existing observation of power law distributions in various kinds of real sports tournaments therefore suggests that the rules of those games are constructed in such a way that it is possible to understand the games in terms of the contestants' inherent characteristics of competitiveness.

  14. Altered Reward Circuitry in the Norepinephrine Transporter Knockout Mouse

    PubMed Central

    Hall, F. Scott; Uhl, George R.; Bearer, Elaine L.; Jacobs, Russell E.

    2013-01-01

    Synaptic levels of the monoamine neurotransmitters dopamine, serotonin, and norepinephrine are modulated by their respective plasma membrane transporters, albeit with a few exceptions. Monoamine transporters remove monoamines from the synaptic cleft and thus influence the degree and duration of signaling. Abnormal concentrations of these neuronal transmitters are implicated in a number of neurological and psychiatric disorders, including addiction, depression, and attention deficit/hyperactivity disorder. This work concentrates on the norepinephrine transporter (NET), using a battery of in vivo magnetic resonance imaging techniques and histological correlates to probe the effects of genetic deletion of the norepinephrine transporter on brain metabolism, anatomy and functional connectivity. MRS recorded in the striatum of NET knockout mice indicated a lower concentration of NAA that correlates with histological observations of subtle dysmorphisms in the striatum and internal capsule. As with DAT and SERT knockout mice, we detected minimal structural alterations in NET knockout mice by tensor-based morphometric analysis. In contrast, longitudinal imaging after stereotaxic prefrontal cortical injection of manganese, an established neuronal circuitry tracer, revealed that the reward circuit in the NET knockout mouse is biased toward anterior portions of the brain. This is similar to previous results observed for the dopamine transporter (DAT) knockout mouse, but dissimilar from work with serotonin transporter (SERT) knockout mice where Mn2+ tracings extended to more posterior structures than in wildtype animals. These observations correlate with behavioral studies indicating that SERT knockout mice display anxiety-like phenotypes, while NET knockouts and to a lesser extent DAT knockout mice display antidepressant-like phenotypic features. Thus, the mainly anterior activity detected with manganese-enhanced MRI in the DAT and NET knockout mice is likely indicative of

  15. Altered reward circuitry in the norepinephrine transporter knockout mouse.

    PubMed

    Gallagher, Joseph J; Zhang, Xiaowei; Hall, F Scott; Uhl, George R; Bearer, Elaine L; Jacobs, Russell E

    2013-01-01

    Synaptic levels of the monoamine neurotransmitters dopamine, serotonin, and norepinephrine are modulated by their respective plasma membrane transporters, albeit with a few exceptions. Monoamine transporters remove monoamines from the synaptic cleft and thus influence the degree and duration of signaling. Abnormal concentrations of these neuronal transmitters are implicated in a number of neurological and psychiatric disorders, including addiction, depression, and attention deficit/hyperactivity disorder. This work concentrates on the norepinephrine transporter (NET), using a battery of in vivo magnetic resonance imaging techniques and histological correlates to probe the effects of genetic deletion of the norepinephrine transporter on brain metabolism, anatomy and functional connectivity. MRS recorded in the striatum of NET knockout mice indicated a lower concentration of NAA that correlates with histological observations of subtle dysmorphisms in the striatum and internal capsule. As with DAT and SERT knockout mice, we detected minimal structural alterations in NET knockout mice by tensor-based morphometric analysis. In contrast, longitudinal imaging after stereotaxic prefrontal cortical injection of manganese, an established neuronal circuitry tracer, revealed that the reward circuit in the NET knockout mouse is biased toward anterior portions of the brain. This is similar to previous results observed for the dopamine transporter (DAT) knockout mouse, but dissimilar from work with serotonin transporter (SERT) knockout mice where Mn(2+) tracings extended to more posterior structures than in wildtype animals. These observations correlate with behavioral studies indicating that SERT knockout mice display anxiety-like phenotypes, while NET knockouts and to a lesser extent DAT knockout mice display antidepressant-like phenotypic features. Thus, the mainly anterior activity detected with manganese-enhanced MRI in the DAT and NET knockout mice is likely indicative of

  16. 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

  17. Proteomic Analysis of Loricrin Knockout Mouse Epidermis.

    PubMed

    Rice, Robert H; Durbin-Johnson, Blythe P; Ishitsuka, Yosuke; Salemi, Michelle; Phinney, Brett S; Rocke, David M; Roop, Dennis R

    2016-08-01

    The crosslinked envelope of the mammalian epidermal corneocyte serves as a scaffold for assembly of the lipid barrier of the epidermis. Thus, deficient envelope crosslinking by keratinocyte transglutaminase (TGM1) is a major cause of the human autosomal recessive congenital ichthyoses characterized by barrier defects. Expectations that loss of some envelope protein components would also confer an ichthyosis phenotype have been difficult to demonstrate. To help rationalize this observation, the protein profile of epidermis from loricrin knockout mice has been compared to that of wild type. Despite the mild phenotype of the knockout, some 40 proteins were incorporated into envelope material to significantly different extents compared to those of wild type. Nearly half were also incorporated to similarly altered extents into the disulfide bonded keratin network of the corneocyte. The results suggest that loss of loricrin alters their incorporation into envelopes as a consequence of protein-protein interactions during cell maturation. Mass spectrometric protein profiling revealed that keratin 1, keratin 10, and loricrin are prominent envelope components and that dozens of other proteins are also components. This finding helps rationalize the potential formation of functional envelopes, despite loss of a single component, due to the availability of many alternative transglutaminase substrates. PMID:27418529

  18. Tissue-specific knockouts of steroidogenic factor 1.

    PubMed

    Zhao, Liping; Bakke, Marit; Hanley, Neil A; Majdic, Gregor; Stallings, Nancy R; Jeyasuria, Pancharatnam; Parker, Keith L

    2004-02-27

    Targeted gene disruption has produced knockout (KO) mice globally deficient in the orphan nuclear receptor steroidogenic factor 1 (SF-1). These SF-1 KO mice lacked adrenal glands and gonads, and also had impaired expression of gonadotropins in pituitary gonadotropes and marked structural abnormalities of the ventromedial hypothalamic nucleus (VMH). To define the roles of SF-1 within discrete sites of the hypothalamic-pituitary-steroidogenic organ axis, we have sought to make tissue-specific SF-1 KO mice (as reviewed here). We first used adrenal transplants to restore adrenal function in global SF-1 KO mice, providing a physiological form of a "VMH-specific" KO to study the roles of SF-1 in weight regulation. These adrenal-transplanted SF-1 KO mice became obese due to decreased locomotor activity, providing a novel model of hypothalamic obesity. Mice with a pituitary-specific KO of SF-1 mediated by the Cre-loxP recombination strategy exhibited hypogonadotropic hypogonadism, revealing essential roles of SF-1 in pituitary function in vivo. Ongoing studies seek to inactivate SF-1 in the brain or specific gonadal cell types, thereby defining its roles in development and function at these sites. In addition, we review our use of bacterial artificial chromosome transgenesis to develop a fluorescent marker for cells that express SF-1.

  19. 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

  20. CRISPR-Mediated Triple Knockout of SLAMF1, SLAMF5 and SLAMF6 Supports Positive Signaling Roles in NKT Cell Development

    PubMed Central

    Huang, Bonnie; Gomez-Rodriguez, Julio; Preite, Silvia; Garrett, Lisa J.; Harper, Ursula L.; Schwartzberg, Pamela L.

    2016-01-01

    The SLAM family receptors contribute to diverse aspects of lymphocyte biology and signal via the small adaptor molecule SAP. Mutations affecting SAP lead to X-linked lymphoproliferative syndrome Type 1, a severe immunodysregulation characterized by fulminant mononucleosis, dysgammaglobulinemia, and lymphoproliferation/lymphomas. Patients and mice having mutations affecting SAP also lack germinal centers due to a defect in T:B cell interactions and are devoid of invariant NKT (iNKT) cells. However, which and how SLAM family members contribute to these phenotypes remains uncertain. Three SLAM family members: SLAMF1, SLAMF5 and SLAMF6, are highly expressed on T follicular helper cells and germinal center B cells. SLAMF1 and SLAMF6 are also implicated in iNKT development. Although individual receptor knockout mice have limited iNKT and germinal center phenotypes compared to SAP knockout mice, the generation of multi-receptor knockout mice has been challenging, due to the genomic linkage of the genes encoding SLAM family members. Here, we used Cas9/CRISPR-based mutagenesis to generate mutations simultaneously in Slamf1, Slamf5 and Slamf6. Genetic disruption of all three receptors in triple-knockout mice (TKO) did not grossly affect conventional T or B cell development and led to mild defects in germinal center formation post-immunization. However, the TKO worsened defects in iNKT cells development seen in SLAMF6 single gene-targeted mice, supporting data on positive signaling and potential redundancy between these receptors. PMID:27258160

  1. Hunting Viral Receptors Using Haploid Cells

    PubMed Central

    Pillay, Sirika; Carette, Jan E.

    2016-01-01

    Viruses have evolved intricate mechanisms to gain entry into the host cell. Identification of critical receptors has enabled insights into virus particle internalization, host and tissue tropism, and viral pathogenesis. In this review we discuss the most commonly employed methods for virus receptor discovery, specifically highlighting the use of forward genetic screens in human haploid cells. The ability to generate true knockout alleles at high saturation provides a sensitive means to study virus-host interactions. As an example, haploid genetic screens identified the lysosomal proteins, NPC1 and LAMP1, as intracellular receptors for Ebola virus and Lassa virus, respectively. From these studies emerges the notion that receptor usage by these viruses is highly dynamic involving a programmed switch from cell surface receptor to intracellular receptor. Broad application of genetic knockout approaches will chart functional landscapes of receptors and endocytic pathways hijacked by viruses. PMID:26958914

  2. 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.

  3. Universal statistics of the knockout tournament

    PubMed Central

    Baek, Seung Ki; Yi, Il Gu; Park, Hye Jin; Kim, Beom Jun

    2013-01-01

    We study statistics of the knockout tournament, where only the winner of a fixture progresses to the next. We assign a real number called competitiveness to each contestant and find that the resulting distribution of prize money follows a power law with an exponent close to unity if the competitiveness is a stable quantity and a decisive factor to win a match. Otherwise, the distribution is found narrow. The existing observation of power law distributions in various kinds of real sports tournaments therefore suggests that the rules of those games are constructed in such a way that it is possible to understand the games in terms of the contestants' inherent characteristics of competitiveness. PMID:24217406

  4. Universal statistics of the knockout tournament.

    PubMed

    Baek, Seung Ki; Yi, Il Gu; Park, Hye Jin; Kim, Beom Jun

    2013-01-01

    We study statistics of the knockout tournament, where only the winner of a fixture progresses to the next. We assign a real number called competitiveness to each contestant and find that the resulting distribution of prize money follows a power law with an exponent close to unity if the competitiveness is a stable quantity and a decisive factor to win a match. Otherwise, the distribution is found narrow. The existing observation of power law distributions in various kinds of real sports tournaments therefore suggests that the rules of those games are constructed in such a way that it is possible to understand the games in terms of the contestants' inherent characteristics of competitiveness. PMID:24217406

  5. 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

  6. 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.

  7. Positron emission tomography and functional characterization of a complete PBR/TSPO knockout.

    PubMed

    Banati, Richard B; Middleton, Ryan J; Chan, Ronald; Hatty, Claire R; Kam, Winnie Wai-Ying; Quin, Candice; Graeber, Manuel B; Parmar, Arvind; Zahra, David; Callaghan, Paul; Fok, Sandra; Howell, Nicholas R; Gregoire, Marie; Szabo, Alexander; Pham, Tien; Davis, Emma; Liu, Guo-Jun

    2014-11-19

    The evolutionarily conserved peripheral benzodiazepine receptor (PBR), or 18-kDa translocator protein (TSPO), is thought to be essential for cholesterol transport and steroidogenesis, and thus life. TSPO has been proposed as a biomarker of neuroinflammation and a new drug target in neurological diseases ranging from Alzheimer's disease to anxiety. Here we show that global C57BL/6-Tspo(tm1GuWu(GuwiyangWurra))-knockout mice are viable with normal growth, lifespan, cholesterol transport, blood pregnenolone concentration, protoporphyrin IX metabolism, fertility and behaviour. However, while the activation of microglia after neuronal injury appears to be unimpaired, microglia from (GuwiyangWurra)TSPO knockouts produce significantly less ATP, suggesting reduced metabolic activity. Using the isoquinoline PK11195, the ligand originally used for the pharmacological and structural characterization of the PBR/TSPO, and the imidazopyridines CLINDE and PBR111, we demonstrate the utility of (GuwiyangWurra)TSPO knockouts to provide robust data on drug specificity and selectivity, both in vitro and in vivo, as well as the mechanism of action of putative TSPO-targeting drugs.

  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. Positron emission tomography and functional characterization of a complete PBR/TSPO knockout

    PubMed Central

    Banati, Richard B.; Middleton, Ryan J.; Chan, Ronald; Hatty, Claire R.; Wai-Ying Kam, Winnie; Quin, Candice; Graeber, Manuel B.; Parmar, Arvind; Zahra, David; Callaghan, Paul; Fok, Sandra; Howell, Nicholas R.; Gregoire, Marie; Szabo, Alexander; Pham, Tien; Davis, Emma; Liu, Guo-Jun

    2014-01-01

    The evolutionarily conserved peripheral benzodiazepine receptor (PBR), or 18-kDa translocator protein (TSPO), is thought to be essential for cholesterol transport and steroidogenesis, and thus life. TSPO has been proposed as a biomarker of neuroinflammation and a new drug target in neurological diseases ranging from Alzheimer’s disease to anxiety. Here we show that global C57BL/6-Tspotm1GuWu(GuwiyangWurra)-knockout mice are viable with normal growth, lifespan, cholesterol transport, blood pregnenolone concentration, protoporphyrin IX metabolism, fertility and behaviour. However, while the activation of microglia after neuronal injury appears to be unimpaired, microglia from GuwiyangWurraTSPO knockouts produce significantly less ATP, suggesting reduced metabolic activity. Using the isoquinoline PK11195, the ligand originally used for the pharmacological and structural characterization of the PBR/TSPO, and the imidazopyridines CLINDE and PBR111, we demonstrate the utility of GuwiyangWurraTSPO knockouts to provide robust data on drug specificity and selectivity, both in vitro and in vivo, as well as the mechanism of action of putative TSPO-targeting drugs. PMID:25406832

  10. mRNA expression profile of serotonin receptor subtypes and distribution of serotonergic terminations in marmoset brain

    PubMed Central

    Shukla, Rammohan; Watakabe, Akiya; Yamamori, Tetsuo

    2014-01-01

    To better understand serotonin function in the primate brain, we examined the mRNA expression patterns of all the 13 members of the serotonin receptor (5HTR) family, by in situ hybridization (ISH) and the distribution of serotonergic terminations by serotonin transporter (SERT) protein immunohistochemical analysis. Ten of the 13 5HTRs showed significant mRNA expressions in the marmoset brain. Our study shows several new features of the organization of serotonergic systems in the marmoset brain. (1) The thalamus expressed only a limited number of receptor subtypes compared with the cortex, hippocampus, and other subcortical regions. (2) In the cortex, there are layer-selective and area-selective mRNA expressions of 5HTRs. (3) Highly localized mRNA expressions of 5HT1F and 5HT3A were observed. (4) There was a conspicuous overlap of the mRNA expressions of receptor subtypes known to have somatodendritic localization of receptor proteins with dense serotonergic terminations in the visual cortex, the central lateral (CL) nucleus of the thalamus, the presubiculum, and the medial mammillary nucleus of the hypothalamus. This suggests a high correlation between serotonin availability and receptor expression at these locations. (5) The 5HTRs show differences in mRNA expression pattern between the marmoset and mouse cortices whereas the patterns of both the species were much similar in the hippocampus. We discuss the possible roles of 5HTRs in the marmoset brain revealed by the analysis of their overall mRNA expression patterns. PMID:24904298

  11. 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.

  12. Development of a Markerless Knockout Method for Actinobacillus succinogenes

    PubMed Central

    Joshi, Rajasi V.; Schindler, Bryan D.; McPherson, Nikolas R.; Tiwari, Kanupriya

    2014-01-01

    Actinobacillus succinogenes is one of the best natural succinate-producing organisms, but it still needs engineering to further increase succinate yield and productivity. In this study, we developed a markerless knockout method for A. succinogenes using natural transformation or electroporation. The Escherichia coli isocitrate dehydrogenase gene with flanking flippase recognition target sites was used as the positive selection marker, making use of A. succinogenes's auxotrophy for glutamate to select for growth on isocitrate. The Saccharomyces cerevisiae flippase recombinase (Flp) was used to remove the selection marker, allowing its reuse. Finally, the plasmid expressing flp was cured using acridine orange. We demonstrate that at least two consecutive deletions can be introduced into the same strain using this approach, that no more than a total of 1 kb of DNA is needed on each side of the selection cassette to protect from exonuclease activity during transformation, and that no more than 200 bp of homologous DNA is needed on each side for efficient recombination. We also demonstrate that electroporation can be used as an alternative transformation method to obtain knockout mutants and that an enriched defined medium can be used for direct selection of knockout mutants on agar plates with high efficiency. Single-knockout mutants of the fumarate reductase and of the pyruvate formate lyase-encoding genes were obtained using this knockout strategy. Double-knockout mutants were also obtained by deleting the citrate lyase-, β-galactosidase-, and aconitase-encoding genes in the pyruvate formate lyase knockout mutant strain. PMID:24610845

  13. Activation of human 5-hydroxytryptamine type 3 receptors via an allosteric transmembrane site.

    PubMed

    Lansdell, Stuart J; Sathyaprakash, Chaitra; Doward, Anne; Millar, Neil S

    2015-01-01

    In common with other members of the Cys-loop family of pentameric ligand-gated ion channels, 5-hydroxytryptamine type 3 receptors (5-HT3Rs) are activated by the binding of a neurotransmitter to an extracellular orthosteric site, located at the interface of two adjacent receptor subunits. In addition, a variety of compounds have been identified that modulate agonist-evoked responses of 5-HT3Rs, and other Cys-loop receptors, by binding to distinct allosteric sites. In this study, we examined the pharmacological effects of a group of monoterpene compounds on recombinant 5-HT3Rs expressed in Xenopus oocytes. Two phenolic monoterpenes (carvacrol and thymol) display allosteric agonist activity on human homomeric 5-HT3ARs (64 ± 7% and 80 ± 4% of the maximum response evoked by the endogenous orthosteric agonist 5-HT, respectively). In addition, at lower concentrations, where agonist effects are less apparent, carvacrol and thymol act as potentiators of responses evoked by submaximal concentrations of 5-HT. By contrast, carvacrol and thymol have no agonist or potentiating activity on the closely related mouse 5-HT3ARs. Using subunit chimeras containing regions of the human and mouse 5-HT3A subunits, and by use of site-directed mutagenesis, we have identified transmembrane amino acids that either abolish the agonist activity of carvacrol and thymol on human 5-HT3ARs or are able to confer this property on mouse 5-HT3ARs. By contrast, these mutations have no significant effect on orthosteric activation of 5-HT3ARs by 5-HT. We conclude that 5-HT3ARs can be activated by the binding of ligands to an allosteric transmembrane site, a conclusion that is supported by computer docking studies. PMID:25338672

  14. Human Knockout Carriers: Dead, Diseased, Healthy, or Improved?

    PubMed Central

    Narasimhan, Vagheesh M.; Xue, Yali; Tyler-Smith, Chris

    2016-01-01

    Whole-genome and whole-exome sequence data from large numbers of individuals reveal that we all carry many variants predicted to inactivate genes (knockouts). This discovery raises questions about the phenotypic consequences of these knockouts and potentially allows us to study human gene function through the investigation of homozygous loss-of-function carriers. Here, we discuss strategies, recent results, and future prospects for large-scale human knockout studies. We examine their relevance to studying gene function, population genetics, and importantly, the implications for accurate clinical interpretations. PMID:26988438

  15. 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.

  16. AMPK: Lessons from transgenic and knockout animals

    PubMed Central

    Viollet, Benoit; Athea, Yoni; Mounier, Remi; Guigas, Bruno; Zarrinpashneh, Elham; Horman, Sandrine; Lantier, Louise; Hebrard, Sophie; Devin-Leclerc, Jocelyne; Beauloye, Christophe; Foretz, Marc; Andreelli, Fabrizio; Ventura-Clapier, Renee; Bertrand, Luc

    2009-01-01

    AMP-activated protein kinase (AMPK), a phylogenetically conserved serine/threonine protein kinase, has been proposed to function as a ‘fuel gauge’ to monitor cellular energy status in response to nutritional environmental variations. AMPK system is a regulator of energy balance that, once activated by low energy status, switches on ATP-producing catabolic pathways (such as fatty acid oxidation and glycolysis), and switches off ATP-consuming anabolic pathways (such as lipogenesis), both by short-term effect on phosphorylation of regulatory proteins and by long-term effect on gene expression. Numerous observations obtained with pharmacological activators and agents that deplete intracellular ATP have been supportive of AMPK playing a role in the control of energy metabolism but none of these studies have provided conclusive evidence. Relatively recent developments in our understanding of precisely how AMPK complexes might operate to control energy metabolism is due in part to the development of transgenic and knockout mouse models. Although there are inevitable caveats with genetic models, some important findings have emerged. In the present review, we discuss recent findings obtained from animal models with inhibition or activation of AMPK signaling pathway. PMID:19273052

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. Combination of cell culture assays and knockout mouse analyses for the study of opioid partial agonism.

    PubMed

    Ide, Soichiro; Minami, Masabumi; Sora, Ichiro; Ikeda, Kazutaka

    2010-01-01

    Nonselective opioid partial agonists, such as buprenorphine, butorphanol, and pentazocine, have been widely used as analgesics and for anti-addiction therapy. However, the precise molecular mechanisms underlying the therapeutic and rewarding effects of these drugs have not been clearly delineated. Recent success in developing mu-opioid receptor knockout (MOP-KO) mice has elucidated the molecular mechanisms underlying the effects of morphine and other opioids. We have revealed the in vivo roles of MOPs in the effects of opioid partial agonists by using MOP-KO mice for behavioral tests (e.g., several kinds of antinociceptive tests for analgesic effects, conditioned place preference test for dependence). The combination of the cell culture assays using cDNA for mu, delta, and kappa opioid receptors and the behavioral tests using MOP-KO mice has provided novel theories on the molecular mechanisms underlying the effects of opioid ligands, especially opioid partial agonists. PMID:20336435

  2. Interactions of the opioid and cannabinoid systems in reward: Insights from knockout studies.

    PubMed

    Befort, Katia

    2015-01-01

    The opioid system consists of three receptors, mu, delta, and kappa, which are activated by endogenous opioid peptides (enkephalins, endorphins, and dynorphins). The endogenous cannabinoid system comprises lipid neuromodulators (endocannabinoids), enzymes for their synthesis and their degradation and two well-characterized receptors, cannabinoid receptors CB1 and CB2. These systems play a major role in the control of pain as well as in mood regulation, reward processing and the development of addiction. Both opioid and cannabinoid receptors are coupled to G proteins and are expressed throughout the brain reinforcement circuitry. Extending classical pharmacology, research using genetically modified mice has provided important progress in the identification of the specific contribution of each component of these endogenous systems in vivo on reward process. This review will summarize available genetic tools and our present knowledge on the consequences of gene knockout on reinforced behaviors in both systems, with a focus on their potential interactions. A better understanding of opioid-cannabinoid interactions may provide novel strategies for therapies in addicted individuals. PMID:25698968

  3. 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.

  4. Cathepsin K knockout alleviates aging-induced cardiac dysfunction.

    PubMed

    Hua, Yinan; Robinson, Timothy J; Cao, Yongtao; Shi, Guo-Ping; Ren, Jun; Nair, Sreejayan

    2015-06-01

    Aging is a major risk factor for cardiovascular disease. It has previously been shown that protein levels of cathepsin K, a lysosomal cysteine protease, are elevated in the failing heart and that genetic ablation of cathepsin K protects against pressure overload-induced cardiac hypertrophy and contractile dysfunction. Here we test the hypothesis that cathepsin K knockout alleviates age-dependent decline in cardiac function. Cardiac geometry, contractile function, intracellular Ca(2+) properties, and cardiomyocyte apoptosis were evaluated using echocardiography, fura-2 technique, immunohistochemistry, Western blot and TUNEL staining, respectively. Aged (24-month-old) mice exhibited significant cardiac remodeling (enlarged chamber size, wall thickness, myocyte cross-sectional area, and fibrosis), decreased cardiac contractility, prolonged relengthening along with compromised intracellular Ca(2+) release compared to young (6-month-old) mice, which were attenuated in the cathepsin K knockout mice. Cellular markers of senescence, including cardiac lipofuscin, p21 and p16, were lower in the aged-cathepsin K knockout mice compared to their wild-type counterpart. Mechanistically, cathepsin K knockout mice attenuated an age-induced increase in cardiomyocyte apoptosis and nuclear translocation of mitochondrial apoptosis-inducing factor (AIF). In cultured H9c2 cells, doxorubicin stimulated premature senescence and apoptosis. Silencing of cathepsin K blocked the doxorubicin-induced translocation of AIF from the mitochondria to the nuclei. Collectively, these results suggest that cathepsin K knockout attenuates age-related decline in cardiac function via suppressing caspase-dependent and caspase-independent apoptosis. PMID:25692548

  5. Cathepsin K knockout alleviates aging-induced cardiac dysfunction

    PubMed Central

    Hua, Yinan; Robinson, Timothy J; Cao, Yongtao; Shi, Guo-Ping; Ren, Jun; Nair, Sreejayan

    2015-01-01

    Aging is a major risk factor for cardiovascular disease. It has previously been shown that protein levels of cathepsin K, a lysosomal cysteine protease, are elevated in the failing heart and that genetic ablation of cathepsin K protects against pressure overload-induced cardiac hypertrophy and contractile dysfunction. Here we test the hypothesis that cathepsin K knockout alleviates age-dependent decline in cardiac function. Cardiac geometry, contractile function, intracellular Ca2+ properties, and cardiomyocyte apoptosis were evaluated using echocardiography, fura-2 technique, immunohistochemistry, Western blot and TUNEL staining, respectively. Aged (24-month-old) mice exhibited significant cardiac remodeling (enlarged chamber size, wall thickness, myocyte cross-sectional area, and fibrosis), decreased cardiac contractility, prolonged relengthening along with compromised intracellular Ca2+ release compared to young (6-month-old) mice, which were attenuated in the cathepsin K knockout mice. Cellular markers of senescence, including cardiac lipofuscin, p21 and p16, were lower in the aged-cathepsin K knockout mice compared to their wild-type counterpart. Mechanistically, cathepsin K knockout mice attenuated an age-induced increase in cardiomyocyte apoptosis and nuclear translocation of mitochondrial apoptosis-inducing factor (AIF). In cultured H9c2 cells, doxorubicin stimulated premature senescence and apoptosis. Silencing of cathepsin K blocked the doxorubicin-induced translocation of AIF from the mitochondria to the nuclei. Collectively, these results suggest that cathepsin K knockout attenuates age-related decline in cardiac function via suppressing caspase-dependent and caspase-independent apoptosis. PMID:25692548

  6. 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

  7. Cathepsin K knockout alleviates aging-induced cardiac dysfunction.

    PubMed

    Hua, Yinan; Robinson, Timothy J; Cao, Yongtao; Shi, Guo-Ping; Ren, Jun; Nair, Sreejayan

    2015-06-01

    Aging is a major risk factor for cardiovascular disease. It has previously been shown that protein levels of cathepsin K, a lysosomal cysteine protease, are elevated in the failing heart and that genetic ablation of cathepsin K protects against pressure overload-induced cardiac hypertrophy and contractile dysfunction. Here we test the hypothesis that cathepsin K knockout alleviates age-dependent decline in cardiac function. Cardiac geometry, contractile function, intracellular Ca(2+) properties, and cardiomyocyte apoptosis were evaluated using echocardiography, fura-2 technique, immunohistochemistry, Western blot and TUNEL staining, respectively. Aged (24-month-old) mice exhibited significant cardiac remodeling (enlarged chamber size, wall thickness, myocyte cross-sectional area, and fibrosis), decreased cardiac contractility, prolonged relengthening along with compromised intracellular Ca(2+) release compared to young (6-month-old) mice, which were attenuated in the cathepsin K knockout mice. Cellular markers of senescence, including cardiac lipofuscin, p21 and p16, were lower in the aged-cathepsin K knockout mice compared to their wild-type counterpart. Mechanistically, cathepsin K knockout mice attenuated an age-induced increase in cardiomyocyte apoptosis and nuclear translocation of mitochondrial apoptosis-inducing factor (AIF). In cultured H9c2 cells, doxorubicin stimulated premature senescence and apoptosis. Silencing of cathepsin K blocked the doxorubicin-induced translocation of AIF from the mitochondria to the nuclei. Collectively, these results suggest that cathepsin K knockout attenuates age-related decline in cardiac function via suppressing caspase-dependent and caspase-independent apoptosis.

  8. GPR55 and the vascular receptors for cannabinoids.

    PubMed

    Hiley, C R; Kaup, S S

    2007-11-01

    CB1 and CB2 receptors mediate most responses to cannabinoids but not some of the cardiovascular actions of endocannabinoids such as anandamide and virodhamine, or those of some synthetic agents, like abnormal cannabidiol (abn-cbd). These agents induce vasorelaxation which is antagonised by rimonabant but only at high concentrations relative to those required to block CB1 receptors. Vasorelaxation to anandamide is sensitive to Pertussis toxin (though that to abn-cbd is not), and so is thought to be mediated by a G protein-coupled receptor through Gi/o. An orphan receptor, GPR55, apparently a cannabinoid receptor, is activated by abn-cbd, but is not the receptor mediating vasorelaxation to this agent, as the response persists in vessels from GPR55 knockout mice. However, the activity of anandamide in GPR55 knockout mice is not yet reported and so the role of GPR55 as a cannabinoid receptor mediating vascular responses has yet to be finalised.

  9. Knockout silkworms reveal a dispensable role for juvenile hormones in holometabolous life cycle

    PubMed Central

    Daimon, Takaaki; Uchibori, Miwa; Nakao, Hajime; Sezutsu, Hideki; Shinoda, Tetsuro

    2015-01-01

    Insect juvenile hormones (JHs) prevent precocious metamorphosis and allow larvae to undergo multiple rounds of status quo molts. However, the roles of JHs during the embryonic and very early larval stages have not been fully understood. We generated and characterized knockout silkworms (Bombyx mori) with null mutations in JH biosynthesis or JH receptor genes using genome-editing tools. We found that embryonic growth and morphogenesis are largely independent of JHs in Bombyx and that, even in the absence of JHs or JH signaling, pupal characters are not formed in first- or second-instar larvae, and precocious metamorphosis is induced after the second instar at the earliest. We also show by mosaic analysis that a pupal specifier gene broad, which is dramatically up-regulated in the late stage of the last larval instar, is essential for pupal commitment in the epidermis. Importantly, the mRNA expression level of broad, which is thought to be repressed by JHs, remained at very low basal levels during the early larval instars of JH-deficient or JH signaling-deficient knockouts. Therefore, our study suggests that the long-accepted paradigm that JHs maintain the juvenile status throughout larval life should be revised because the larval status can be maintained by a JH-independent mechanism in very early larval instars. We propose that the lack of competence for metamorphosis during the early larval stages may result from the absence of an unidentified broad-inducing factor, i.e., a competence factor. PMID:26195792

  10. Adipose-specific knockout of SEIPIN/BSCL2 results in progressive lipodystrophy.

    PubMed

    Liu, Lu; Jiang, Qingqing; Wang, Xuhong; Zhang, Yuxi; Lin, Ruby C Y; Lam, Sin Man; Shui, Guanghou; Zhou, Linkang; Li, Peng; Wang, Yuhui; Cui, Xin; Gao, Mingming; Zhang, Ling; Lv, Ying; Xu, Guoheng; Liu, George; Zhao, Dong; Yang, Hongyuan

    2014-07-01

    Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2) is the most severe form of human lipodystrophy, characterized by an almost complete loss of adipose tissue and severe insulin resistance. BSCL2 is caused by loss-of-function mutations in the BSCL2/SEIPIN gene, which is upregulated during adipogenesis and abundantly expressed in the adipose tissue. The physiological function of SEIPIN in mature adipocytes, however, remains to be elucidated. Here, we generated adipose-specific Seipin knockout (ASKO) mice, which exhibit adipocyte hypertrophy with enlarged lipid droplets, reduced lipolysis, adipose tissue inflammation, progressive loss of white and brown adipose tissue, insulin resistance, and hepatic steatosis. Lipidomic and microarray analyses revealed accumulation/imbalance of lipid species, including ceramides, in ASKO adipose tissue as well as increased endoplasmic reticulum stress. Interestingly, the ASKO mice almost completely phenocopy the fat-specific peroxisome proliferator-activated receptor-γ (Pparγ) knockout (FKO-γ) mice. Rosiglitazone treatment significantly improved a number of metabolic parameters of the ASKO mice, including insulin sensitivity. Our results therefore demonstrate a critical role of SEIPIN in maintaining lipid homeostasis and function of adipocytes and reveal an intimate relationship between SEIPIN and PPAR-γ.

  11. A rat knockout model implicates TRPC4 in visceral pain sensation.

    PubMed

    Westlund, K N; Zhang, L P; Ma, F; Nesemeier, R; Ruiz, J C; Ostertag, E M; Crawford, J S; Babinski, K; Marcinkiewicz, M M

    2014-03-14

    Acute and chronic pain resulting from injury, surgery, or disease afflicts >100 million Americans each year, having a severe impact on mood, mental health, and quality of life. The lack of structural and functional information for most ion channels, many of which play key roles in the detection and transmission of noxious stimuli, means that there remain unidentified therapeutic targets for pain management. This study focuses on the transient receptor potential canonical subfamily 4 (TRPC4) ion channel, which is involved in the tissue-specific and stimulus-dependent regulation of intracellular Ca²⁺ signaling. Rats with a transposon-mediated TRPC4-knockout mutation displayed tolerance to visceral pain induced by colonic mustard oil (MO) exposure, but not somatic or neuropathic pain stimuli. Moreover, wild-type rats treated with a selective TRPC4 antagonist (ML-204) prior to MO exposure mimicked the behavioral responses observed in TRPC4-knockout rats. Significantly, ML-204 inhibited visceral pain-related behavior in a dose-dependent manner without noticeable adverse effects. These data provide evidence that TRPC4 is required for detection and/or transmission of colonic MO visceral pain sensation. In the future, inhibitors of TRPC4 signaling may provide a highly promising path for the development of first-in-class therapeutics for this visceral pain, which may have fewer side effects and less addictive potential than opioid derivatives.

  12. Knockout silkworms reveal a dispensable role for juvenile hormones in holometabolous life cycle.

    PubMed

    Daimon, Takaaki; Uchibori, Miwa; Nakao, Hajime; Sezutsu, Hideki; Shinoda, Tetsuro

    2015-08-01

    Insect juvenile hormones (JHs) prevent precocious metamorphosis and allow larvae to undergo multiple rounds of status quo molts. However, the roles of JHs during the embryonic and very early larval stages have not been fully understood. We generated and characterized knockout silkworms (Bombyx mori) with null mutations in JH biosynthesis or JH receptor genes using genome-editing tools. We found that embryonic growth and morphogenesis are largely independent of JHs in Bombyx and that, even in the absence of JHs or JH signaling, pupal characters are not formed in first- or second-instar larvae, and precocious metamorphosis is induced after the second instar at the earliest. We also show by mosaic analysis that a pupal specifier gene broad, which is dramatically up-regulated in the late stage of the last larval instar, is essential for pupal commitment in the epidermis. Importantly, the mRNA expression level of broad, which is thought to be repressed by JHs, remained at very low basal levels during the early larval instars of JH-deficient or JH signaling-deficient knockouts. Therefore, our study suggests that the long-accepted paradigm that JHs maintain the juvenile status throughout larval life should be revised because the larval status can be maintained by a JH-independent mechanism in very early larval instars. We propose that the lack of competence for metamorphosis during the early larval stages may result from the absence of an unidentified broad-inducing factor, i.e., a competence factor. PMID:26195792

  13. 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

  14. 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

  15. Multi-Functional OCT Enables Longitudinal Study of Retinal Changes in a VLDLR Knockout Mouse Model

    PubMed Central

    Fialová, Stanislava; Himmel, Tanja; Glösmann, Martin; Lengheimer, Theresia; Harper, Danielle J.; Plasenzotti, Roberto; Pircher, Michael; Hitzenberger, Christoph K.; Baumann, Bernhard

    2016-01-01

    We present a multi-functional optical coherence tomography (OCT) imaging approach to study retinal changes in the very-low-density-lipoprotein-receptor (VLDLR) knockout mouse model with a threefold contrast. In the retinas of VLDLR knockout mice spontaneous retinal-chorodoidal neovascularizations form, having an appearance similar to choroidal and retinal neovascularizations (CNV and RNV) in neovascular age-related macular degeneration (AMD) or retinal angiomatous proliferation (RAP). For this longitudinal study, the mice were imaged every 4 to 6 weeks starting with an age of 4 weeks and following up to the age of 11 months. Significant retinal changes were identified by the multi-functional imaging approach offering a threefold contrast: reflectivity, polarization sensitivity (PS) and motion contrast based OCT angiography (OCTA). By use of this intrinsic contrast, the long-term development of neovascularizations was studied and associated processes, such as the migration of melanin pigments or retinal-choroidal anastomosis, were assessed in vivo. Furthermore, the in vivo imaging results were validated with histological sections at the endpoint of the experiment. Multi-functional OCT proves as a powerful tool for longitudinal retinal studies in preclinical research of ophthalmic diseases. Intrinsic contrast offered by the functional extensions of OCT might help to describe regulative processes in genetic animal models and potentially deepen the understanding of the pathogenesis of retinal diseases such as wet AMD. PMID:27711217

  16. 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

  17. The 5-HT[subscript 3A] Receptor Is Essential for Fear Extinction

    ERIC Educational Resources Information Center

    Kondo, Makoto; Nakamura, Yukiko; Ishida, Yusuke; Yamada, Takahiro; Shimada, Shoichi

    2014-01-01

    The 5-HT [subscript 3] receptor, the only ionotropic 5-HT receptor, is expressed in limbic regions, including the hippocampus, amygdala, and cortex. However, it is not known whether it has a role in fear memory processes. Analysis of 5-HT [subscript 3A] receptor knockout mice in fear conditioning paradigms revealed that the 5-HT [subscript 3A]…

  18. 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.

  19. Generation of myometrium-specific Bmal1 knockout mice for parturition analysis.

    PubMed

    Ratajczak, Christine K; Asada, Minoru; Allen, Gregg C; McMahon, Douglas G; Muglia, Lisa M; Smith, Donté; Bhattacharyya, Sandip; Muglia, Louis J

    2012-01-01

    Human and rodent studies indicate a role for circadian rhythmicity and associated clock gene expression in supporting normal parturition. The importance of clock gene expression in tissues besides the suprachiasmatic nucleus is emerging. Here, a Bmal1 conditional knockout mouse line and a novel Cre transgenic mouse line were used to examine the role of myometrial Bmal1 in parturition. Ninety-two percent (22/24) of control females but only 64% (14/22) of females with disrupted myometrial Bmal1 completed parturition during the expected time window of 5p.m. on Day 19 through to 9a.m. on Day 19.5 of gestation. However, neither serum progesterone levels nor uterine transcript expression of the contractile-associated proteins Connexin43 and Oxytocin receptor differed between females with disrupted myometrial Bmal1 and controls during late gestation. The data indicate a role for myometrial Bmal1 in maintaining normal time of day of parturition.

  20. 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

  1. Selective Photoreceptor Gene Knock-out Reveals a Regulatory Role for the Growth Behavior of Pseudomonas syringae.

    PubMed

    Shah, Rashmi; Pathak, Gopal; Drepper, Thomas; Gärtner, Wolfgang

    2016-07-01

    The plant pathogen Pseudomonas syringae (Ps) is a well-established model organism for bacterial infection of plants. The genome sequences of two pathovars, pv. syringae and pv. tomato, revealed one gene encoding a blue and two genes encoding red/far red light-sensing photoreceptors. Continuing former molecular characterization of the photoreceptor proteins, we here report selective photoreceptor gene disruption for pv. tomato aiming at identification of potentially regulatory functions of these photoreceptors. Transformation of Ps cells with linear DNA constructs yielded interposon mutations of the corresponding genes. Cell growth studies of the generated photoreceptor knock-out mutants revealed their role in light-dependent regulation of cell growth and motility. Disruption of the blue-light (BL) receptor gene caused a growth deregulation, in line with an observed increased virulence of this mutant (Moriconi et al., Plant J., 2013, 76, 322). Bacterial phytochrome-1 (BphP1) deletion mutant caused unaltered cell growth, but a stronger swarming capacity. Inactivation of its ortholog, BphP2, however, caused reduced growth and remarkably altered dendritic swarming behavior. Combined knock-out of both bacteriophytochromes reproduced the swarming pattern observed for the BphP2 mutant alone. A triple knock-out mutant showed a growth rate between that of the BL (deregulation) and the phytochrome-2 mutant (growth reduction).

  2. Selective Photoreceptor Gene Knock-out Reveals a Regulatory Role for the Growth Behavior of Pseudomonas syringae.

    PubMed

    Shah, Rashmi; Pathak, Gopal; Drepper, Thomas; Gärtner, Wolfgang

    2016-07-01

    The plant pathogen Pseudomonas syringae (Ps) is a well-established model organism for bacterial infection of plants. The genome sequences of two pathovars, pv. syringae and pv. tomato, revealed one gene encoding a blue and two genes encoding red/far red light-sensing photoreceptors. Continuing former molecular characterization of the photoreceptor proteins, we here report selective photoreceptor gene disruption for pv. tomato aiming at identification of potentially regulatory functions of these photoreceptors. Transformation of Ps cells with linear DNA constructs yielded interposon mutations of the corresponding genes. Cell growth studies of the generated photoreceptor knock-out mutants revealed their role in light-dependent regulation of cell growth and motility. Disruption of the blue-light (BL) receptor gene caused a growth deregulation, in line with an observed increased virulence of this mutant (Moriconi et al., Plant J., 2013, 76, 322). Bacterial phytochrome-1 (BphP1) deletion mutant caused unaltered cell growth, but a stronger swarming capacity. Inactivation of its ortholog, BphP2, however, caused reduced growth and remarkably altered dendritic swarming behavior. Combined knock-out of both bacteriophytochromes reproduced the swarming pattern observed for the BphP2 mutant alone. A triple knock-out mutant showed a growth rate between that of the BL (deregulation) and the phytochrome-2 mutant (growth reduction). PMID:27289014

  3. Functional Chimeras of GLIC Obtained by Adding the Intracellular Domain of Anion- and Cation-Conducting Cys-Loop Receptors.

    PubMed

    Mnatsakanyan, Nelli; Nishtala, Sita Nirupama; Pandhare, Akash; Fiori, Mariana C; Goyal, Raman; Pauwels, Jonathan E; Navetta, Andrew F; Ahrorov, Afzal; Jansen, Michaela

    2015-04-28

    Pentameric ligand-gated ion channels (pLGICs), also called Cys-loop receptors in eukaryotic superfamily members, play diverse roles in neurotransmission and serve as primary targets for many therapeutic drugs. Structural studies of full-length eukaryotic pLGICs have been challenging because of glycosylation, large size, pentameric assembly, and hydrophobicity. X-ray structures of prokaryotic pLGICs, including the Gloeobacter violaceus LGIC (GLIC) and the Erwinia chrysanthemi LGIC (ELIC), and truncated eukaryotic pLGICs have significantly improved and complemented the understanding of structural details previously obtained with acetylcholine-binding protein and Torpedo nicotinic acetylcholine receptors. Prokaryotic pLGICs share their overall structural features with eukaryotic pLGICs for the ligand-binding extracellular and channel-lining transmembrane domains. The large intracellular domain (ICD) is present only in eukaryotic members and is characterized by a low level of sequence conservation and significant variability in length (50-250 amino acids), making the ICD a potential target for the modulation of specific pLGIC subunits. None of the structures includes a complete ICD. Here, we created chimeras by adding the ICD of cation-conducting (nAChR-α7) and anion-conducting (GABAρ1, Glyα1) eukaryotic homopentamer-forming pLGICs to GLIC. GLIC-ICD chimeras assemble into pentamers to form proton-gated channels, as does the parent GLIC. Additionally, the sensitivity of the chimeras toward modulation of functional maturation by chaperone protein RIC-3 is preserved as in those of the parent eukaryotic channels. For a previously described GLIC-5HT3A-ICD chimera, we now provide evidence of its successful large-scale expression and purification to homogeneity. Overall, the chimeras provide valuable tools for functional and structural studies of eukaryotic pLGIC ICDs. PMID:25861708

  4. 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

  5. A Knockout Mutation of a Constitutive GPCR in Tetrahymena Decreases Both G-Protein Activity and Chemoattraction

    PubMed Central

    Lampert, Thomas J.; Coleman, Kevin D.; Hennessey, Todd M.

    2011-01-01

    Although G-protein coupled receptors (GPCRs) are a common element in many chemosensory transduction pathways in eukaryotic cells, no GPCR or regulated G-protein activity has yet been shown in any ciliate. To study the possible role for a GPCR in the chemoresponses of the ciliate Tetrahymena, we have generated a number of macronuclear gene knockouts of putative GPCRs found in the Tetrahymena Genome database. One of these knockout mutants, called G6, is a complete knockout of a gene that we call GPCR6 (TTHERM_00925490). Based on sequence comparisons, the Gpcr6p protein belongs to the Rhodopsin Family of GPCRs. Notably, Gpcr6p shares highest amino acid sequence homologies to GPCRs from Paramecium and several plants. One of the phenotypes of the G6 mutant is a decreased responsiveness to the depolarizing ions Ba2+ and K+, suggesting a decrease in basal excitability (decrease in Ca2+ channel activity). The other major phenotype of G6 is a loss of chemoattraction to lysophosphatidic acid (LPA) and proteose peptone (PP), two known chemoattractants in Tetrahymena. Using microsomal [35S]GTPγS binding assays, we found that wild-type (CU427) have a prominent basal G-protein activity. This activity is decreased to the same level by pertussis toxin (a G-protein inhibitor), addition of chemoattractants, or the G6 mutant. Since the basal G-protein activity is decreased by the GPCR6 knockout, it is likely that this gene codes for a constitutively active GPCR in Tetrahymena. We propose that chemoattractants like LPA and PP cause attraction in Tetrahymena by decreasing the basal G-protein stimulating activity of Gpcr6p. This leads to decreased excitability in wild-type and longer runs of smooth forward swimming (less interrupted by direction changes) towards the attractant. Therefore, these attractants may work as inverse agonists through the constitutively active Gpcr6p coupled to a pertussis-sensitive G-protein. PMID:22140501

  6. Role of estrogen receptor-α on food demand elasticity.

    PubMed

    Minervini, Vanessa; Rowland, Neil E; Robertson, Kimberly L; Foster, Thomas C

    2015-05-01

    Estrogens have been shown to have an inhibitory effect on food intake under free-feeding conditions, yet the effects of estrogens on food-maintained operant responding have been studied to a much lesser extent and, thus, are not well understood. Therefore, the purpose of the present experiment was to use a behavioral economics paradigm to assess differences in demand elasticity between mice with knockout of the estrogen receptor subtype α, knockout of subtype β, and their wild type controls. The mice responded in a closed economy, and the price of food was increased by increasing the fixed-ratio response requirement every four sessions. Overall, we found that mice with the knockout of receptor subtype α had the most elastic demand functions. Therefore, under these conditions, estrogens increased food seeking via activation of the receptor subtype α. The results were inconsistent with those reported by previous studies that employed free-feeding conditions. PMID:25869426

  7. Role of estrogen receptor-α on food demand elasticity.

    PubMed

    Minervini, Vanessa; Rowland, Neil E; Robertson, Kimberly L; Foster, Thomas C

    2015-05-01

    Estrogens have been shown to have an inhibitory effect on food intake under free-feeding conditions, yet the effects of estrogens on food-maintained operant responding have been studied to a much lesser extent and, thus, are not well understood. Therefore, the purpose of the present experiment was to use a behavioral economics paradigm to assess differences in demand elasticity between mice with knockout of the estrogen receptor subtype α, knockout of subtype β, and their wild type controls. The mice responded in a closed economy, and the price of food was increased by increasing the fixed-ratio response requirement every four sessions. Overall, we found that mice with the knockout of receptor subtype α had the most elastic demand functions. Therefore, under these conditions, estrogens increased food seeking via activation of the receptor subtype α. The results were inconsistent with those reported by previous studies that employed free-feeding conditions.

  8. GABA(B) receptors in neuroendocrine regulation.

    PubMed

    Lux-Lantos, Victoria A; Bianchi, María S; Catalano, Paolo N; Libertun, Carlos

    2008-09-01

    Gamma-amino butyric acid (GABA), in addition to being a metabolic intermediate and the main inhibitory neurotransmitter in the synaptic cleft, is postulated as a neurohormone, a paracrine signaling molecule, and a trophic factor. It acts through pre- and post-synaptic receptors, named GABA(A) and GABA(C) (ionotropic receptors) and GABA(B) (metabotropic receptor). Here we reviewed the participation of GABA(B) receptors in the regulation of the hypothalamic-pituitary-gonadal axis, using physiological, biochemical, and pharmacological approaches in rats, as well as in GABA(B1) knock-out mice, that lack functional GABA(B) receptors. Our general conclusion indicates that GABA(B )receptors participate in the regulation of pituitary hormone secretion acting both in the central nervous system and directly on the gland. PRL and gonadotropin axes are affected by GABA(B) receptor activation, as demonstrated in the rat and also in the GABA(B1) knock-out mouse. In addition, hypothalamic and pituitary GABA(B) receptor expression is modulated by steroid hormones. GABA participation in the brain control of pituitary secretion through GABA(B) receptors depends on physiological conditions, being age and sex critical factors.These results indicate that patients receiving GABA(B) agonists/antagonists should be monitored for possible endocrine side effects.

  9. The GABAA receptor is an FMRP target with therapeutic potential in fragile X syndrome

    PubMed Central

    Braat, Sien; D'Hulst, Charlotte; Heulens, Inge; De Rubeis, Silvia; Mientjes, Edwin; Nelson, David L; Willemsen, Rob; Bagni, Claudia; Van Dam, Debby; De Deyn, Peter P; Kooy, R Frank

    2015-01-01

    Previous research indicates that the GABAAergic system is involved in the pathophysiology of the fragile X syndrome, a frequent form of inherited intellectual disability and associated with autism spectrum disorder. However, the molecular mechanism underlying GABAAergic deficits has remained largely unknown. Here, we demonstrate reduced mRNA expression of GABAA receptor subunits in the cortex and cerebellum of young Fmr1 knockout mice. In addition, we show that the previously reported underexpression of specific subunits of the GABAA receptor can be corrected in YAC transgenic rescue mice, containing the full-length human FMR1 gene in an Fmr1 knockout background. Moreover, we demonstrate that FMRP directly binds several GABAA receptor mRNAs. Finally, positive allosteric modulation of GABAA receptors with the neurosteroid ganaxolone can modulate specific behaviors in Fmr1 knockout mice, emphasizing the therapeutic potential of the receptor. PMID:25790165

  10. 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'.

  11. 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

  12. 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

  13. Pauli blocking and medium effects in nucleon knockout reactions

    SciTech Connect

    Bertulani, C. A.; De Conti, C.

    2010-06-15

    We study medium modifications of the nucleon-nucleon (NN) cross sections and their influence on the nucleon knockout reactions. Using the eikonal approximation, we compare the results obtained with free NN cross sections with those obtained with a purely geometrical treatment of Pauli blocking and with NN obtained with more elaborated Dirac-Bruecker methods. The medium effects are parametrized in terms of the baryon density. We focus on symmetric nuclear matter, although the geometrical Pauli blocking also allows for the treatment of asymmetric nuclear matter. It is shown that medium effects can change the nucleon knockout cross sections and momentum distributions up to 10% in the energy range E{sub lab}=50-300 MeV/nucleon. The effect is more evident in reactions involving halo nuclei.

  14. Targeted gene knockout in chickens mediated by TALENs.

    PubMed

    Park, Tae Sub; Lee, Hong Jo; Kim, Ki Hyun; Kim, Jin-Soo; Han, Jae Yong

    2014-09-01

    Genetically modified animals are used for industrial applications as well as scientific research, and studies on these animals contribute to a better understanding of biological mechanisms. Gene targeting techniques have been developed to edit specific gene loci in the genome, but the conventional strategy of homologous recombination with a gene-targeted vector has low efficiency and many technical complications. Here, we generated specific gene knockout chickens through the use of transcription activator-like effector nuclease (TALEN)-mediated gene targeting. In this study, we accomplished targeted knockout of the ovalbumin (OV) gene in the chicken primordial germ cells, and OV gene mutant offspring were generated through test-cross analysis. TALENs successfully induced nucleotide deletion mutations of ORF shifts, resulting in loss of chicken OV gene function. Our results demonstrate that the TALEN technique used in the chicken primordial germ cell line is a powerful strategy to create specific genome-edited chickens safely for practical applications. PMID:25139993

  15. Knockout driven reactions in complex molecules and their clusters

    NASA Astrophysics Data System (ADS)

    Gatchell, Michael; Zettergren, Henning

    2016-08-01

    Energetic ions lose some of their kinetic energy when interacting with electrons or nuclei in matter. Here, we discuss combined experimental and theoretical studies on such impulse driven reactions in polycyclic aromatic hydrocarbons (PAHs), fullerenes, and pure or mixed clusters of these molecules. These studies show that the nature of excitation is important for how complex molecular systems respond to ion/atom impact. Rutherford-like nuclear scattering processes may lead to prompt atom knockout and formation of highly reactive fragments, while heating of the molecular electron clouds in general lead to formation of more stable and less reactive fragments. In this topical review, we focus on recent studies of knockout driven reactions, and present new calculations of the angular dependent threshold (displacement) energies for such processes in PAHs. The so-formed fragments may efficiently form covalent bonds with neighboring molecules in clusters. These unique molecular growth processes may be important in astrophysical environments such as low velocity shock waves.

  16. 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

  17. Knock-out models reveal new aquaporin functions.

    PubMed

    Verkman, Alan S

    2009-01-01

    Knockout mice have been informative in the discovery of unexpected biological functions of aquaporins. Knockout mice have confirmed the predicted roles of aquaporins in transepithelial fluid transport, as in the urinary concentrating mechanism and glandular fluid secretion. A less obvious, though predictable role of aquaporins is in tissue swelling under stress, as in the brain in stroke, tumor and infection. Phenotype analysis of aquaporin knockout mice has revealed several unexpected cellular roles of aquaporins whose mechanisms are being elucidated. Aquaporins facilitate cell migration, as seen in aquaporin-dependent tumor angiogenesis and tumor metastasis, by a mechanism that may involve facilitated water transport in lamellipodia of migrating cells. The ' aquaglyceroporins', aquaporins that transport both glycerol and water, regulate glycerol content in epidermis, fat and other tissues, and lead to a multiplicity of interesting consequences of gene disruption including dry skin, resistance to skin carcinogenesis, impaired cell proliferation and altered fat metabolism. An even more surprising role of a mammalian aquaporin is in neural signal transduction in the central nervous system. The many roles of aquaporins might be exploited for clinical benefit by modulation of aquaporin expression/function - as diuretics, and in the treatment of brain swelling, glaucoma, epilepsy, obesity and cancer. PMID:19096787

  18. 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

  19. 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.

  20. 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.

  1. Establishment of ApoE-knockout mouse model of preeclampsia and relevant mechanisms

    PubMed Central

    Sun, Wenjuan; Cui, Baoxia; Hong, Fanzhen; Xu, Yongping

    2016-01-01

    In the present study, we established an ApoE-knockout mouse model of preeclampsia to examine the role of vascular endothelial injury associated with abnormal lipid metabolism in the pathogenesis of preeclampsia. To establish the ApoE-knockout homozygous (ApoE−/−) and heterozygous (ApoE+/−) mouse model, mice were mated with the same genotype and orbital blood on day 19 of conception was collected. The progeny mice were assigned into 3 groups: ApoE−/, ApoE+/− and wild-type (WT) groups. Total cholesterol, triglyceride, low-density and high-density lipoprotein were measured in the serum at the end of conception. During conception, the systolic blood pressure of caudal artery was measured every 4 days. Using bicinchoninic acid protein assay, urinary protein and creatinine ratio was measured with a creatinine kit. We observed the pathological changes of glomerular filtration membrane and macroscopic/microscopic morphological changes of placenta by hematoxylin and eosin (H&E) staining and transmission electron microscope. Take fetal mouse through cesarean section on 19th day, measure the birth weight and placental weight of fetal mouse. Using ELISA we measured the expression levels of toll-like receptor 4 (TLR4) and soluble fms-like tyrosine kinase-1 (sFlt-1). Our results showed that the differences in serum lipid levels were not statistically significant (P>0.05). The mean systolic blood pressure, urinary protein and creatinine in ApoE−/− group were significantly higher than ApoE+/− group and WT group (P<0.05). Thickening and edema of glomerular filtration membrane, capillary thrombosis, significant edema and necrosis of placental villous stroma were observed in ApoE−/− group. No significant change was detected in the ApoE+/− or WT group. The TLR4 and sFlt-1 expression levels in ApoE−/− group were significantly higher than ApoE+/− and WT group (P<0.05). We concluded that ApoE-knockout mouse could simulate the pathologic process of preeclampsia

  2. Establishment of ApoE-knockout mouse model of preeclampsia and relevant mechanisms

    PubMed Central

    Sun, Wenjuan; Cui, Baoxia; Hong, Fanzhen; Xu, Yongping

    2016-01-01

    In the present study, we established an ApoE-knockout mouse model of preeclampsia to examine the role of vascular endothelial injury associated with abnormal lipid metabolism in the pathogenesis of preeclampsia. To establish the ApoE-knockout homozygous (ApoE−/−) and heterozygous (ApoE+/−) mouse model, mice were mated with the same genotype and orbital blood on day 19 of conception was collected. The progeny mice were assigned into 3 groups: ApoE−/, ApoE+/− and wild-type (WT) groups. Total cholesterol, triglyceride, low-density and high-density lipoprotein were measured in the serum at the end of conception. During conception, the systolic blood pressure of caudal artery was measured every 4 days. Using bicinchoninic acid protein assay, urinary protein and creatinine ratio was measured with a creatinine kit. We observed the pathological changes of glomerular filtration membrane and macroscopic/microscopic morphological changes of placenta by hematoxylin and eosin (H&E) staining and transmission electron microscope. Take fetal mouse through cesarean section on 19th day, measure the birth weight and placental weight of fetal mouse. Using ELISA we measured the expression levels of toll-like receptor 4 (TLR4) and soluble fms-like tyrosine kinase-1 (sFlt-1). Our results showed that the differences in serum lipid levels were not statistically significant (P>0.05). The mean systolic blood pressure, urinary protein and creatinine in ApoE−/− group were significantly higher than ApoE+/− group and WT group (P<0.05). Thickening and edema of glomerular filtration membrane, capillary thrombosis, significant edema and necrosis of placental villous stroma were observed in ApoE−/− group. No significant change was detected in the ApoE+/− or WT group. The TLR4 and sFlt-1 expression levels in ApoE−/− group were significantly higher than ApoE+/− and WT group (P<0.05). We concluded that ApoE-knockout mouse could simulate the pathologic process of preeclampsia

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. Transferrin Receptor Controls AMPA Receptor Trafficking Efficiency and Synaptic Plasticity

    PubMed Central

    Liu, Ke; Lei, Run; Li, Qiong; Wang, Xin-Xin; Wu, Qian; An, Peng; Zhang, Jianchao; Zhu, Minyan; Xu, Zhiheng; Hong, Yang; Wang, Fudi; Shen, Ying; Li, Hongchang; Li, Huashun

    2016-01-01

    Transferrin receptor (TFR) is an important iron transporter regulating iron homeostasis and has long been used as a marker for clathrin mediated endocytosis. However, little is known about its additional function other than iron transport in the development of central nervous system (CNS). Here we demonstrate that TFR functions as a regulator to control AMPA receptor trafficking efficiency and synaptic plasticity. The conditional knockout (KO) of TFR in neural progenitor cells causes mice to develop progressive epileptic seizure, and dramatically reduces basal synaptic transmission and long-term potentiation (LTP). We further demonstrate that TFR KO remarkably reduces the binding efficiency of GluR2 to AP2 and subsequently decreases AMPA receptor endocytosis and recycling. Thus, our study reveals that TFR functions as a novel regulator to control AMPA trafficking efficiency and synaptic plasticity. PMID:26880306

  8. Guwiyang Wurra--'Fire Mouse': a global gene knockout model for TSPO/PBR drug development, loss-of-function and mechanisms of compensation studies.

    PubMed

    Middleton, Ryan J; Liu, Guo-Jun; Banati, Richard B

    2015-08-01

    The highly conserved 18-kDa translocator protein (TSPO) or peripheral benzodiazepine receptor (PBR), is being investigated as a diagnostic and therapeutic target for disease conditions ranging from inflammation to neurodegeneration and behavioural illnesses. Many functions have been attributed to TSPO/PBR including a role in the mitochondrial permeability transition pore (MPTP), steroidogenesis and energy metabolism. In this review, we detail the recent developments in determining the physiological role of TSPO/PBR, specifically based on data obtained from the recently generated Tspo knockout mouse models. In addition to defining the role of TSPO/PBR, we also describe the value of Tspo knockout mice in determining the selectivity, specificity and presence of any off-target effects of TSPO/PBR ligands.

  9. The Novel, Nicotinic Alpha7 Receptor Partial Agonist, BMS-933043, Improves Cognition and Sensory Processing in Preclinical Models of Schizophrenia

    PubMed Central

    Bristow, Linda J.; Easton, Amy E.; Li, Yu-Wen; Sivarao, Digavalli V.; Lidge, Regina; Jones, Kelli M.; Post-Munson, Debra; Daly, Christopher; Lodge, Nicholas J.; Gallagher, Lizbeth; Molski, Thaddeus; Pieschl, Richard; Chen, Ping; Hendricson, Adam; Westphal, Ryan; Cook, James; Iwuagwu, Christiana; Morgan, Daniel; Benitex, Yulia; King, Dalton; Macor, John E.; Zaczek, Robert; Olson, Richard

    2016-01-01

    The development of alpha7 nicotinic acetylcholine receptor agonists is considered a promising approach for the treatment of cognitive symptoms in schizophrenia patients. In the present studies we characterized the novel agent, (2R)-N-(6-(1H-imidazol-1-yl)-4-pyrimidinyl)-4'H-spiro[4-azabicyclo[2.2.2]octane-2,5'-[1,3]oxazol]-2'-amine (BMS-933043), in vitro and in rodent models of schizophrenia-like deficits in cognition and sensory processing. BMS-933043 showed potent binding affinity to native rat (Ki = 3.3 nM) and recombinant human alpha7 nicotinic acetylcholine receptors (Ki = 8.1 nM) and agonist activity in a calcium fluorescence assay (EC50 = 23.4 nM) and whole cell voltage clamp electrophysiology (EC50 = 0.14 micromolar (rat) and 0.29 micromolar (human)). BMS-933043 exhibited a partial agonist profile relative to acetylcholine; the relative efficacy for net charge crossing the cell membrane was 67% and 78% at rat and human alpha7 nicotinic acetylcholine receptors respectively. BMS-933043 showed no agonist or antagonist activity at other nicotinic acetylcholine receptor subtypes and was at least 300 fold weaker at binding to and antagonizing human 5-HT3A receptors (Ki = 2,451 nM; IC50 = 8,066 nM). BMS-933043 treatment i) improved 24 hour novel object recognition memory in mice (0.1–10 mg/kg, sc), ii) reversed MK-801-induced deficits in Y maze performance in mice (1–10 mg/kg, sc) and set shift performance in rats (1–10 mg/kg, po) and iii) reduced the number of trials required to complete the extradimensional shift discrimination in neonatal PCP treated rats performing the intra-dimensional/extradimensional set shifting task (0.1–3 mg/kg, po). BMS-933043 also improved auditory gating (0.56–3 mg/kg, sc) and mismatch negativity (0.03–3 mg/kg, sc) in rats treated with S(+)ketamine or neonatal phencyclidine respectively. Given this favorable preclinical profile BMS-933043 was selected for further development to support clinical evaluation in humans. PMID

  10. The Novel, Nicotinic Alpha7 Receptor Partial Agonist, BMS-933043, Improves Cognition and Sensory Processing in Preclinical Models of Schizophrenia.

    PubMed

    Bristow, Linda J; Easton, Amy E; Li, Yu-Wen; Sivarao, Digavalli V; Lidge, Regina; Jones, Kelli M; Post-Munson, Debra; Daly, Christopher; Lodge, Nicholas J; Gallagher, Lizbeth; Molski, Thaddeus; Pieschl, Richard; Chen, Ping; Hendricson, Adam; Westphal, Ryan; Cook, James; Iwuagwu, Christiana; Morgan, Daniel; Benitex, Yulia; King, Dalton; Macor, John E; Zaczek, Robert; Olson, Richard

    2016-01-01

    The development of alpha7 nicotinic acetylcholine receptor agonists is considered a promising approach for the treatment of cognitive symptoms in schizophrenia patients. In the present studies we characterized the novel agent, (2R)-N-(6-(1H-imidazol-1-yl)-4-pyrimidinyl)-4'H-spiro[4-azabicyclo[2.2.2]octane-2,5'-[1,3]oxazol]-2'-amine (BMS-933043), in vitro and in rodent models of schizophrenia-like deficits in cognition and sensory processing. BMS-933043 showed potent binding affinity to native rat (Ki = 3.3 nM) and recombinant human alpha7 nicotinic acetylcholine receptors (Ki = 8.1 nM) and agonist activity in a calcium fluorescence assay (EC50 = 23.4 nM) and whole cell voltage clamp electrophysiology (EC50 = 0.14 micromolar (rat) and 0.29 micromolar (human)). BMS-933043 exhibited a partial agonist profile relative to acetylcholine; the relative efficacy for net charge crossing the cell membrane was 67% and 78% at rat and human alpha7 nicotinic acetylcholine receptors respectively. BMS-933043 showed no agonist or antagonist activity at other nicotinic acetylcholine receptor subtypes and was at least 300 fold weaker at binding to and antagonizing human 5-HT3A receptors (Ki = 2,451 nM; IC50 = 8,066 nM). BMS-933043 treatment i) improved 24 hour novel object recognition memory in mice (0.1-10 mg/kg, sc), ii) reversed MK-801-induced deficits in Y maze performance in mice (1-10 mg/kg, sc) and set shift performance in rats (1-10 mg/kg, po) and iii) reduced the number of trials required to complete the extradimensional shift discrimination in neonatal PCP treated rats performing the intra-dimensional/extradimensional set shifting task (0.1-3 mg/kg, po). BMS-933043 also improved auditory gating (0.56-3 mg/kg, sc) and mismatch negativity (0.03-3 mg/kg, sc) in rats treated with S(+)ketamine or neonatal phencyclidine respectively. Given this favorable preclinical profile BMS-933043 was selected for further development to support clinical evaluation in humans. PMID:27467081

  11. The Novel, Nicotinic Alpha7 Receptor Partial Agonist, BMS-933043, Improves Cognition and Sensory Processing in Preclinical Models of Schizophrenia.

    PubMed

    Bristow, Linda J; Easton, Amy E; Li, Yu-Wen; Sivarao, Digavalli V; Lidge, Regina; Jones, Kelli M; Post-Munson, Debra; Daly, Christopher; Lodge, Nicholas J; Gallagher, Lizbeth; Molski, Thaddeus; Pieschl, Richard; Chen, Ping; Hendricson, Adam; Westphal, Ryan; Cook, James; Iwuagwu, Christiana; Morgan, Daniel; Benitex, Yulia; King, Dalton; Macor, John E; Zaczek, Robert; Olson, Richard

    2016-01-01

    The development of alpha7 nicotinic acetylcholine receptor agonists is considered a promising approach for the treatment of cognitive symptoms in schizophrenia patients. In the present studies we characterized the novel agent, (2R)-N-(6-(1H-imidazol-1-yl)-4-pyrimidinyl)-4'H-spiro[4-azabicyclo[2.2.2]octane-2,5'-[1,3]oxazol]-2'-amine (BMS-933043), in vitro and in rodent models of schizophrenia-like deficits in cognition and sensory processing. BMS-933043 showed potent binding affinity to native rat (Ki = 3.3 nM) and recombinant human alpha7 nicotinic acetylcholine receptors (Ki = 8.1 nM) and agonist activity in a calcium fluorescence assay (EC50 = 23.4 nM) and whole cell voltage clamp electrophysiology (EC50 = 0.14 micromolar (rat) and 0.29 micromolar (human)). BMS-933043 exhibited a partial agonist profile relative to acetylcholine; the relative efficacy for net charge crossing the cell membrane was 67% and 78% at rat and human alpha7 nicotinic acetylcholine receptors respectively. BMS-933043 showed no agonist or antagonist activity at other nicotinic acetylcholine receptor subtypes and was at least 300 fold weaker at binding to and antagonizing human 5-HT3A receptors (Ki = 2,451 nM; IC50 = 8,066 nM). BMS-933043 treatment i) improved 24 hour novel object recognition memory in mice (0.1-10 mg/kg, sc), ii) reversed MK-801-induced deficits in Y maze performance in mice (1-10 mg/kg, sc) and set shift performance in rats (1-10 mg/kg, po) and iii) reduced the number of trials required to complete the extradimensional shift discrimination in neonatal PCP treated rats performing the intra-dimensional/extradimensional set shifting task (0.1-3 mg/kg, po). BMS-933043 also improved auditory gating (0.56-3 mg/kg, sc) and mismatch negativity (0.03-3 mg/kg, sc) in rats treated with S(+)ketamine or neonatal phencyclidine respectively. Given this favorable preclinical profile BMS-933043 was selected for further development to support clinical evaluation in humans.

  12. Assessing the neuronal serotonergic target-based antidepressant stratagem: impact of in vivo interaction studies and knockout models.

    PubMed

    Rajkumar, R; Mahesh, R

    2008-09-01

    Depression remains a challenge in the field of affective neuroscience, despite a steady research progress. Six out of nine basic antidepressant mechanisms rely on serotonin neurotransmitter system. Preclinical studies have demonstrated the significance of serotonin receptors (5-HT(1-3,6,7)), its signal transduction pathways and classical down stream targets (including neurotrophins, neurokinins, other peptides and their receptors) in antidepressant drug action. Serotonergic control of depression embraces the recent molecular requirements such as influence on proliferation, neurogenesis, plasticity, synaptic (re)modeling and transmission in the central nervous system. The present progress report analyses the credibility of each protein as therapeutically relevant target of depression. In vivo interaction studies and knockout models which identified these targets are foreseen to unearth new ligands and help them transform to drug candidates. The importance of the antidepressant assay selection at the preclinical level using salient animal models/assay systems is discussed. Such test batteries would definitely provide antidepressants with faster onset, efficacy in resistant (and co-morbid) types and with least adverse effects. Apart from the selective ligands, only those molecules which bring an overall harmony, by virtue of their affinities to various receptor subtypes, could qualify as effective antidepressants. Synchronised modulation of various serotonergic sub-pathways is the basis for a unique and balanced antidepressant profile, as that of fluoxetine (most exploited antidepressant) and such a profile may be considered as a template for the upcoming antidepressants. In conclusion, 5-HT based multi-targeted antidepressant drug discovery supported by in vivo interaction studies and knockout models is advocated as a strategy to provide classic molecules for clinical trials. PMID:19506722

  13. Pre-Equilibrium Cluster Emission with Pickup and Knockout

    SciTech Connect

    Betak, E.

    2005-05-24

    We present a generalization of the Iwamoto-Harada-Bisplinghoff pre-equilibrium model of light cluster formation and emission, which is enhanced by allowing for possible admixtures of knockout for strongly coupled ejectiles, like {alpha}'s. The model is able to attain the Weisskopf-Ewing formula for compound-nucleus decay at long-time limit; it keeps the philosophy of pre-equilibrium decay during the equilibration stage and it describes the initial phase of a reaction as direct process(es) expressed using the language of the exciton model.

  14. Electron-Induced Neutron Knockout from 4He

    NASA Astrophysics Data System (ADS)

    Misiejuk, A.; Papandreou, Z.; Voutier, E.; Bauer, Th. S.; Blok, H. P.; Boersma, D. J.; den Bok, H. W.; Bruins, E. E.; Farzanpay, F.; Grüner, K.; Hesselink, W. H.; Huber, G. M.; Jans, E.; Kalantar-Nayestanaki, N.; Kasdorp, W.-J.; Konijn, J.; Laget, J.-M.; Lapikás, L.; Lolos, G. J.; Onderwater, G. J.; Pellegrino, A.; Schroevers, R.; Spaltro, C. M.; Starink, R.; van der Steenhoven, G.; Steiger, J. J.; Visschers, J. L.; Willering, H. W.; Yeomans, D. M.

    2002-10-01

    The differential cross section for electron-induced neutron knockout in the reaction 4He(e,e'n)3He has been measured for the first time with a statistical accuracy of 11%. The experiment was performed in quasielastic kinematics at a momentum transfer of 300 MeV/c and in the missing-momentum range of 25-70 MeV/c. The comparison of the data with theoretical calculations shows an impressive increase of the cross section resulting from final state interaction effects. Specifically , the p-n charge-exchange process dominates the cross section in this kinematical regime.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. Conditional knockout of fibronectin abrogates mouse mammary gland lobuloalveolar differentiation

    PubMed Central

    Liu, Keyi; Cheng, Le; Flesken-Nikitin, Andrea; Huang, Lynn; Nikitin, Alexander Y.; Pauli, Bendicht U.

    2010-01-01

    Fibronectin (Fn) plays an important part in the branching morphogenesis of salivary gland, lung, and kidney. Here, we examine the effect of the conditional knockout of Fn in the mammary epithelium [FnMEp−/−] on postnatal mammary gland development, using Cre-loxP mediated gene knockout technology. Our data show that Fn deletion causes a moderate retardation in outgrowth and branching of the ductal tree in 5-week old mice. These defects are partially compensated in virgin 16-week old mice. However, mammary glands consisting of Fn-deficient epithelial cells fail to undergo normal lobuloalveolar differentiation during pregnancy. The severity of lobuloalveolar impairment ranged from lobular hypoplasia to aplasia in some cases and was associated with the amount of Fn protein recovered from these glands. Decreased rates of mammary epithelial cell proliferation accounted for delayed ductal outgrowth in virgin and lack of alveologenesis in pregnant FnMEp−/− mice. Concomitant decreased expression of integrin β1 (Itgb1) and lack of autophosphorylation of focal adhesion kinase (Fak) suggest that this pathology might, at least in part, be mediated by disruption of the Fn/Itgb1/Fak signaling pathway. PMID:20624380

  20. 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

  1. 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

  2. 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

  3. 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

  4. RAG1/2 knockout pigs with severe combined immunodeficiency.

    PubMed

    Huang, Jiao; Guo, Xiaogang; Fan, Nana; Song, Jun; Zhao, Bentian; Ouyang, Zhen; Liu, Zhaoming; Zhao, Yu; Yan, Quanmei; Yi, Xiaoling; Schambach, Axel; Frampton, Jon; Esteban, Miguel A; Yang, Dongshan; Yang, Huaqiang; Lai, Liangxue

    2014-08-01

    Pigs share many physiological, biochemical, and anatomical similarities with humans and have emerged as valuable large animal models for biomedical research. Considering the advantages in immune system resemblance, suitable size, and longevity for clinical practical and monitoring purpose, SCID pigs bearing dysfunctional RAG could serve as important experimental tools for regenerative medicine, allograft and xenograft transplantation, and reconstitution experiments related to the immune system. In this study, we report the generation and phenotypic characterization of RAG1 and RAG2 knockout pigs using transcription activator-like effector nucleases. Porcine fetal fibroblasts were genetically engineered using transcription activator-like effector nucleases and then used to provide donor nuclei for somatic cell nuclear transfer. We obtained 27 live cloned piglets; among these piglets, 9 were targeted with biallelic mutations in RAG1, 3 were targeted with biallelic mutations in RAG2, and 10 were targeted with a monoallelic mutation in RAG2. Piglets with biallelic mutations in either RAG1 or RAG2 exhibited hypoplasia of immune organs, failed to perform V(D)J rearrangement, and lost mature B and T cells. These immunodeficient RAG1/2 knockout pigs are promising tools for biomedical and translational research. PMID:24973446

  5. Evaluation of high efficiency gene knockout strategies for Trypanosoma cruzi

    PubMed Central

    2009-01-01

    Background Trypanosoma cruzi, a kinetoplastid protozoan parasite that causes Chagas disease, infects approximately 15 million people in Central and South America. In contrast to the substantial in silico studies of the T. cruzi genome, transcriptome, and proteome, only a few genes have been experimentally characterized and validated, mainly due to the lack of facile methods for gene manipulation needed for reverse genetic studies. Current strategies for gene disruption in T. cruzi are tedious and time consuming. In this study we have compared the conventional multi-step cloning technique with two knockout strategies that have been proven to work in other organisms, one-step-PCR- and Multisite Gateway-based systems. Results While the one-step-PCR strategy was found to be the fastest method for production of knockout constructs, it does not efficiently target genes of interest using gene-specific sequences of less than 80 nucleotides. Alternatively, the Multisite Gateway based approach is less time-consuming than conventional methods and is able to efficiently and reproducibly delete target genes. Conclusion Using the Multisite Gateway strategy, we have rapidly produced constructs that successfully produce specific gene deletions in epimastigotes of T. cruzi. This methodology should greatly facilitate reverse genetic studies in T. cruzi. PMID:19432966

  6. 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

  7. Interleukin-15 affects serotonin system and exerts antidepressive effects through IL15Rα receptor

    PubMed Central

    Wu, Xiaojun; Hsuchou, Hung; Kastin, Abba J.; He, Yi; Khan, Reas S.; Stone, Kirsten P.; Cash, Michael S.; Pan, Weihong

    2010-01-01

    Summary Contrary to the reduction of depressive-like behavior observed in several strains of cytokine receptor knockout mice, mice lacking the specific receptor for interleukin (IL)-15 showed increased immobility in tail suspension and modified forced swimming tests. There was also a reduction in social interactions. The hippocampus of the IL15Rα knockout mice had decreased mRNA for 5-HT1A, increased mRNA for 5-HT2C, and region-specific changes of serotonin reuptake transporter (SERT) immunoreactivity. Fluoxetine (the classic antidepressant Prozac, which inhibits 5-HT2C and SERT) reduced the immobility of the IL15Rα knockout mice in comparison with their pretreatment baseline. Together with the unchanged performance of the IL15Rα knockout mice on the rotarod, this response to fluoxetine indicates that the immobility reflects depression. Wildtype mice responded to IL15 treatment with improvement of immobility induced by forced swimming, whereas the knockout mice failed to respond. Thus, the cognate IL15 receptor is necessary for the antidepressive activity of IL15. In ex-vivo studies, IL15 decreased synaptosomal uptake of 5-HT, and modulated the expression of 5-HT2C and SERT in cultured neurons in a dose- and time-dependent manner. Thus, the effect of IL15 on serotonin transmission may underlie the depressive-like behavior of IL15Rα knockout mice. We speculate that IL15 is essential to maintain neurochemical homeostasis and thereby plays a role in preventing neuropsychiatric symptoms. PMID:20724079

  8. Polyhydramnios in Lrp4 knockout mice with bilateral kidney agenesis: Defects in the pathways of amniotic fluid clearance.

    PubMed

    Tanahashi, Hiroshi; Tian, Qing-Bao; Hara, Yoshinobu; Sakagami, Hiroyuki; Endo, Shogo; Suzuki, Tatsuo

    2016-01-01

    Amniotic fluid volume during mid-to-late gestation depends mainly on the urine excretion from the foetal kidneys and partly on the fluid secretion from the foetal lungs during foetal breathing-like movements. Urine is necessary for foetal breathing-like movements, which is critical for foetal lung development. Bilateral renal agenesis and/or obstruction of the urinary tract lead to oligohydramnios, which causes infant death within a short period after birth due to pulmonary hypoplasia. Lrp4, which functions as an agrin receptor, is essential for the formation of neuromuscular junctions. Herein, we report novel phenotypes of Lrp4 knockout (Lrp4(-/-)) mice. Most Lrp4(-/-) foetuses showed unilateral or bilateral kidney agenesis, and Lrp4 knockout resulted in polyhydramnios. The loss of Lrp4 compromised foetal swallowing and breathing-like movements and downregulated the expression of aquaporin-9 in the foetal membrane and aquaporin-1 in the placenta, which possibly affected the amniotic fluid clearance. These results suggest that amniotic fluid removal was compromised in Lrp4(-/-) foetuses, resulting in polyhydramnios despite the impairment of urine production. Our findings indicate that amniotic fluid removal plays an essential role in regulating the amniotic fluid volume.

  9. 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.

  10. Polyhydramnios in Lrp4 knockout mice with bilateral kidney agenesis: Defects in the pathways of amniotic fluid clearance

    PubMed Central

    Tanahashi, Hiroshi; Tian, Qing-Bao; Hara, Yoshinobu; Sakagami, Hiroyuki; Endo, Shogo; Suzuki, Tatsuo

    2016-01-01

    Amniotic fluid volume during mid-to-late gestation depends mainly on the urine excretion from the foetal kidneys and partly on the fluid secretion from the foetal lungs during foetal breathing-like movements. Urine is necessary for foetal breathing-like movements, which is critical for foetal lung development. Bilateral renal agenesis and/or obstruction of the urinary tract lead to oligohydramnios, which causes infant death within a short period after birth due to pulmonary hypoplasia. Lrp4, which functions as an agrin receptor, is essential for the formation of neuromuscular junctions. Herein, we report novel phenotypes of Lrp4 knockout (Lrp4−/−) mice. Most Lrp4−/− foetuses showed unilateral or bilateral kidney agenesis, and Lrp4 knockout resulted in polyhydramnios. The loss of Lrp4 compromised foetal swallowing and breathing-like movements and downregulated the expression of aquaporin-9 in the foetal membrane and aquaporin-1 in the placenta, which possibly affected the amniotic fluid clearance. These results suggest that amniotic fluid removal was compromised in Lrp4−/− foetuses, resulting in polyhydramnios despite the impairment of urine production. Our findings indicate that amniotic fluid removal plays an essential role in regulating the amniotic fluid volume. PMID:26847765

  11. Adult Conditional Knockout of PGC-1α Leads to Loss of Dopamine Neurons

    PubMed Central

    Jiang, Haisong; Zhang, Shuran; Karuppagounder, Senthilkumar; Xu, Jinchong; Pletnikova, Olga; Troncoso, Juan C.; Pirooznia, Shelia; Andrabi, Shaida A.

    2016-01-01

    Parkinson’s disease (PD) is a chronic progressive neurodegenerative disorder. Recent studies have implicated a role for peroxisome proliferator-activated receptor γ coactivator protein-1α (PGC-1α) in PD and in animal or cellular models of PD. The role of PGC-1α in the function and survival of substantia nigra pars compacta (SNpc) dopamine neurons is not clear. Here we find that there are four different PGC-1α isoforms expressed in SH-SY5Y cells, and these four isoforms are expressed across subregions of mouse brain. Adult conditional PGC-1α knock-out mice show a significant loss of dopaminergic neurons that is accompanied by a reduction of dopamine in the striatum. In human PD postmortem tissue from the SNpc, there is a reduction of PGC-1α isoforms and mitochondria markers. Our findings suggest that all four isoforms of PGC-1α are required for the proper expression of mitochondrial proteins in SNpc DA neurons and that PGC-1α is essential for SNpc DA neuronal survival, possibly through the maintenance of mitochondrial function. PMID:27622213

  12. Adult Conditional Knockout of PGC-1α Leads to Loss of Dopamine Neurons.

    PubMed

    Jiang, Haisong; Kang, Sung-Ung; Zhang, Shuran; Karuppagounder, Senthilkumar; Xu, Jinchong; Lee, Yong-Kyu; Kang, Bong-Gu; Lee, Yunjong; Zhang, Jianmin; Pletnikova, Olga; Troncoso, Juan C; Pirooznia, Shelia; Andrabi, Shaida A; Dawson, Valina L; Dawson, Ted M

    2016-01-01

    Parkinson's disease (PD) is a chronic progressive neurodegenerative disorder. Recent studies have implicated a role for peroxisome proliferator-activated receptor γ coactivator protein-1α (PGC-1α) in PD and in animal or cellular models of PD. The role of PGC-1α in the function and survival of substantia nigra pars compacta (SNpc) dopamine neurons is not clear. Here we find that there are four different PGC-1α isoforms expressed in SH-SY5Y cells, and these four isoforms are expressed across subregions of mouse brain. Adult conditional PGC-1α knock-out mice show a significant loss of dopaminergic neurons that is accompanied by a reduction of dopamine in the striatum. In human PD postmortem tissue from the SNpc, there is a reduction of PGC-1α isoforms and mitochondria markers. Our findings suggest that all four isoforms of PGC-1α are required for the proper expression of mitochondrial proteins in SNpc DA neurons and that PGC-1α is essential for SNpc DA neuronal survival, possibly through the maintenance of mitochondrial function. PMID:27622213

  13. 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

  14. 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

  15. Common Mechanisms for Calorie Restriction and AC5 Knockout Models of Longevity

    PubMed Central

    Yan, Lin; Park, Ji Yeon; Dillinger, Jean-Guillaume; De Lorenzo, Mariana S.; Yuan, Chujun; Lai, Lo; Wang, Chunbo; Ho, David; Tian, Bin; Stanley, William C; Auwerx, Johan; Vatner, Dorothy E.; Vatner, Stephen F.

    2012-01-01

    Summary Adenylyl cyclase type 5 knockout mice (AC5 KO) live longer and are stress resistant, similar to calorie restriction (CR). AC5 KO mice eat more, but actually weigh less and accumulate less fat compared to WT mice. CR applied to AC5 KO result in rapid decrease in body weight, metabolic deterioration and death. These data suggest that despite restricted food intake in CR, but augmented food intake in AC5 KO, the two models affect longevity and metabolism similarly. To determine shared molecular mechanisms, mRNA expression was examined genome-wide for brain, heart, skeletal muscle and liver. Significantly more genes were regulated commonly rather than oppositely in all the tissues in both models, indicating commonality between AC5 KO and CR. Gene Ontology analysis identified many significantly regulated, tissue-specific pathways shared by the two models, including sensory perception in heart and brain, muscle function in skeletal muscle, and lipid metabolism in liver. Moreover, when comparing gene expression changes in the heart under stress, the glutathione regulatory pathway was consistently upregulated in the longevity models but downregulated with stress. In addition, AC5 and CR shared changes in genes and proteins involved in the regulation of longevity and stress resistance, including Sirt1, ApoD and olfactory receptors in both young and intermediate age mice. Thus, the similarly regulated genes and pathways in AC5 KO and CR, particularly related to the metabolic phenotype, suggest a unified theory for longevity and stress resistance. PMID:23020244

  16. Adult Conditional Knockout of PGC-1α Leads to Loss of Dopamine Neurons

    PubMed Central

    Jiang, Haisong; Zhang, Shuran; Karuppagounder, Senthilkumar; Xu, Jinchong; Pletnikova, Olga; Troncoso, Juan C.; Pirooznia, Shelia; Andrabi, Shaida A.

    2016-01-01

    Parkinson’s disease (PD) is a chronic progressive neurodegenerative disorder. Recent studies have implicated a role for peroxisome proliferator-activated receptor γ coactivator protein-1α (PGC-1α) in PD and in animal or cellular models of PD. The role of PGC-1α in the function and survival of substantia nigra pars compacta (SNpc) dopamine neurons is not clear. Here we find that there are four different PGC-1α isoforms expressed in SH-SY5Y cells, and these four isoforms are expressed across subregions of mouse brain. Adult conditional PGC-1α knock-out mice show a significant loss of dopaminergic neurons that is accompanied by a reduction of dopamine in the striatum. In human PD postmortem tissue from the SNpc, there is a reduction of PGC-1α isoforms and mitochondria markers. Our findings suggest that all four isoforms of PGC-1α are required for the proper expression of mitochondrial proteins in SNpc DA neurons and that PGC-1α is essential for SNpc DA neuronal survival, possibly through the maintenance of mitochondrial function.

  17. 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

  18. 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

  19. 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.

  20. 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

  1. SRA Gene Knockout Protects against Diet-induced Obesity and Improves Glucose Tolerance*

    PubMed Central

    Liu, Shannon; Sheng, Liang; Miao, Hongzhi; Saunders, Thomas L.; MacDougald, Ormond A.; Koenig, Ronald J.; Xu, Bin

    2014-01-01

    We have recently shown that the non-coding RNA, steroid receptor RNA activator (SRA), functions as a transcriptional coactivator of PPARγ and promotes adipocyte differentiation in vitro. To assess SRA function in vivo, we have generated a whole mouse Sra1 gene knock-out (SRA−/−). Here, we show that the Sra1 gene is an important regulator of adipose tissue mass and function. SRA is expressed at a higher level in adipose tissue than other organs in wild type mice. SRA−/− mice are resistant to high fat diet-induced obesity, with decreased fat mass and increased lean content. This lean phenotype of SRA−/− mice is associated with decreased expression of a subset of adipocyte marker genes and reduced plasma TNFα levels. The SRA−/− mice are more insulin sensitive, as evidenced by reduced fasting insulin, and lower blood glucoses in response to IP glucose and insulin. In addition, the livers of SRA−/− mice have fewer lipid droplets after high fat diet feeding, and the expression of lipogenesis-associated genes is decreased. To our knowledge, these data are the first to indicate a functional role for SRA in adipose tissue biology and glucose homeostasis in vivo. PMID:24675075

  2. Increased desensitization of dopamine D₂ receptor-mediated response in the ventral tegmental area in the absence of adenosine A(2A) receptors.

    PubMed

    Al-Hasani, R; Foster, J D; Metaxas, A; Ledent, C; Hourani, S M O; Kitchen, I; Chen, Y

    2011-09-01

    G-protein coupled receptors interact to provide additional regulatory mechanisms for neurotransmitter signaling. Adenosine A(2A) receptors are expressed at a high density in striatal neurons, where they closely interact with dopamine D₂ receptors and modulate effects of dopamine and responses to psychostimulants. A(2A) receptors are expressed at much lower densities in other forebrain neurons but play a more prominent yet opposing role to striatal receptors in response to psychostimulants in mice. It is, therefore, possible that A(2A) receptors expressed at low levels elsewhere in the brain may also regulate neurotransmitter systems and modulate neuronal functions. Dopamine D₂ receptors play an important role in autoinhibition of neuronal firing in dopamine neurons of the ventral tegmental area (VTA) and dopamine release in other brain areas. Here, we examined the effect of A(2A) receptor deletion on D₂ receptor-mediated inhibition of neuronal firing in dopamine neurons in the VTA. Spontaneous activity of dopamine neurons was recorded in midbrain slices, and concentration-dependent effects of the dopamine D₂ receptor agonist, quinpirole, was compared between wild-type and A(2A) knockout mice. The potency of quinpirole applied in single concentrations and the expression of D₂ receptors were not altered in the VTA of the knockout mice. However, quinpirole applied in stepwise escalating concentrations caused significantly reduced maximal inhibition in A(2A) knockout mice, indicating an enhanced agonist-induced desensitization of D₂ receptors in the absence of A(2A) receptors. The A(2A) receptor agonist, CGS21680, did not exert any effect on dopamine neuron firing or response to quinpirole, revealing a novel non-pharmacological interaction between adenosine A(2A) receptors and dopaminergic neurotransmission in midbrain dopamine neurons. Altered D₂ receptor desensitization may result in changes in dopamine neuron firing rate and pattern and dopamine

  3. Endocytic uptake of advanced glycation end products by mouse liver sinusoidal endothelial cells is mediated by a scavenger receptor distinct from the macrophage scavenger receptor class A.

    PubMed Central

    Matsumoto, K; Sano, H; Nagai, R; Suzuki, H; Kodama, T; Yoshida, M; Ueda, S; Smedsrød, B; Horiuchi, S

    2000-01-01

    Previous studies with peritoneal macrophages obtained from macrophage scavenger receptor class A (MSR-A) knock-out mice showed that the endocytic uptake of advanced glycation end products (AGE) by macrophages was mediated mainly by MSR-A. However, it is controversial whether the endocytic uptake of intravenously injected AGE proteins by liver sinusoidal endothelial cells (LECs) is similarly explained by receptor-mediated endocytosis via MSR-A. The present study was conducted to compare the capacity to endocytose AGE proteins in LECs and peritoneal macrophages obtained from MSR-A knock-out and littermate wild-type mice. The endocytic degradation capacity of MSR-A knock-out LECs for AGE-BSA was indistinguishable from that of wild-type LECs, whereas that of MSR-A knock-out peritoneal macrophages for AGE-BSA was decreased to 30% of that in wild-type cells. Similarly, the endocytic degradation of MSR-A knock-out LECs for acetylated low-density lipoprotein (acetyl-LDL) did not differ from that of wild-type LECs, whereas the endocytic degradation of acetyl-LDL by MSR-A knock-out peritoneal macrophages was less than 20% of that in wild-type cells. Furthermore, formaldehyde-treated serum albumin (f-Alb), a ligand known to undergo scavenger-receptor-mediated endocytosis by LECs, was effectively taken up by MSR-A knock-out LECs at a capacity that did not differ from that of wild-type LECs. Moreover, the endocytic uptake of AGE-BSA by LECs was effectively competed for by unlabelled f-Alb or acetyl-LDL. These results indicate that the scavenger-receptor ligands AGE proteins, acetyl-LDL and f-Alb are endocytosed by LECs through a non-MSR-A pathway. PMID:11062078

  4. 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

  5. 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.

  6. 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.

  7. Constitutive Knockout of Kalirin-7 Leads to Increased Rates of Cocaine Self-Administration

    PubMed Central

    Kiraly, Drew D.; Nemirovsky, Natali E.; LaRese, Taylor P.; Tomek, Seven E.; Yahn, Stephanie L.; Olive, M. Foster; Eipper, Betty A.

    2013-01-01

    Kalirin-7 (Kal7) is a Rho-guanine nucleotide exchange factor that is localized in neuronal postsynaptic densities. Kal7 interacts with the NR2B subunit of the NMDA receptor and regulates aspects of dendritic spine dynamics both in vitro and in vivo. Chronic treatment with cocaine increases dendritic spine density in the nucleus accumbens (NAc) of rodents and primates. Kal7 mRNA and protein are upregulated in the NAc following cocaine treatment, and the presence of Kal7 is necessary for the normal proliferation of dendritic spines following cocaine use. Mice that constitutively lack Kal7 [Kalirin-7 knockout mice (Kal7KO)] demonstrate increased locomotor sensitization to cocaine and a decreased place preference for cocaine. Here, using an intravenous cocaine self-administration paradigm, Kal7KO mice exhibit increased administration of cocaine at lower doses as compared with wild-type (Wt) mice. Analyses of mRNA transcript levels from the NAc of mice that self-administered saline or cocaine reveal that larger splice variants of the Kalrn gene are increased by cocaine more dramatically in Kal7KO mice than in Wt mice. Additionally, transcripts encoding the NR2B subunit of the NMDA receptor increased in Wt mice that self-administered cocaine but were unchanged in similarly experienced Kal7KO mice. These findings suggest that Kal7 participates in the reinforcing effects of cocaine, and that Kal7 and cocaine interact to alter the expression of genes related to critical glutamatergic signaling pathways in the NAc. PMID:23894151

  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. Repetitive grooming and sensorimotor abnormalities in an ephrin-A knockout model for Autism Spectrum Disorders

    PubMed Central

    Wurzman, Rachel; Forcelli, Patrick A.; Griffey, Christopher J.; Kromer, Lawrence F.

    2014-01-01

    EphA receptors and ephrin-A ligands play important roles in neural development and synaptic plasticity in brain regions where expression persists into adulthood. Recently, EPHA3 and EPHA7 gene mutations were linked with Autism Spectrum Disorders (ASDs) and developmental neurological delays, respectively. Furthermore, deletions of ephrin-A2 or ephrin-A3, which exhibit high binding affinity for EphA3 and EphA7 receptors, are associated with subtle deficits in learning and memory behavior and abnormalities in dendritic spine morphology in the cortex and hippocampus in mice. To better characterize a potential role for these ligands in ASDs, we performed a comprehensive behavioral characterization of anxiety-like, sensorimotor, learning, and social behaviors in ephrin-A2/-A3 double knockout (DKO) mice. The predominant phenotype in DKO mice was repetitive and self-injurious grooming behaviors such as have been associated with corticostriatal circuit abnormalities in other rodent models of neuropsychiatric disorders. Consistent with ASDs specifically, DKO mice exhibited decreased preference for social interaction in the social approach assay, decreased locomotor activity in the open field, increased prepulse inhibition of acoustic startle, and a shift towards self-directed activity (e.g., grooming) in novel environments, such as marble burying. Although there were no gross deficits in cognitive assays, subtle differences in performance on fear conditioning and in the Morris water maze resembled traits observed in other rodent models of ASD. We therefore conclude that ephrin-A2/-A3 DKO mice have utility as a novel ASD model with an emphasis on sensory abnormalities and restricted, repetitive behavioral symptoms. PMID:25281279

  10. Repetitive grooming and sensorimotor abnormalities in an ephrin-A knockout model for Autism Spectrum Disorders.

    PubMed

    Wurzman, Rachel; Forcelli, Patrick A; Griffey, Christopher J; Kromer, Lawrence F

    2015-02-01

    EphA receptors and ephrin-A ligands play important roles in neural development and synaptic plasticity in brain regions where expression persists into adulthood. Recently, EPHA3 and EPHA7 gene mutations were linked with Autism Spectrum Disorders (ASDs) and developmental neurological delays, respectively. Furthermore, deletions of ephrin-A2 or ephrin-A3, which exhibit high binding affinity for EphA3 and EphA7 receptors, are associated with subtle deficits in learning and memory behavior and abnormalities in dendritic spine morphology in the cortex and hippocampus in mice. To better characterize a potential role for these ligands in ASDs, we performed a comprehensive behavioral characterization of anxiety-like, sensorimotor, learning, and social behaviors in ephrin-A2/-A3 double knockout (DKO) mice. The predominant phenotype in DKO mice was repetitive and self-injurious grooming behaviors such as have been associated with corticostriatal circuit abnormalities in other rodent models of neuropsychiatric disorders. Consistent with ASDs specifically, DKO mice exhibited decreased preference for social interaction in the social approach assay, decreased locomotor activity in the open field, increased prepulse inhibition of acoustic startle, and a shift towards self-directed activity (e.g., grooming) in novel environments, such as marble burying. Although there were no gross deficits in cognitive assays, subtle differences in performance on fear conditioning and in the Morris water maze resembled traits observed in other rodent models of ASD. We therefore conclude that ephrin-A2/-A3 DKO mice have utility as a novel ASD model with an emphasis on sensory abnormalities and restricted, repetitive behavioral symptoms.

  11. Substrate uptake and metabolism are preserved in hypertrophic caveolin-3 knockout hearts

    PubMed Central

    Augustus, Ayanna S.; Buchanan, Jonathan; Addya, Sankar; Rengo, Giuseppe; Pestell, Richard G.; Fortina, Paolo; Koch, Walter J.; Bensadoun, Andre; Abel, E. Dale; Lisanti, Michael P.

    2008-01-01

    Caveolin-3 (Cav3), the primary protein component of caveolae in muscle cells, regulates numerous signaling pathways including insulin receptor signaling and facilitates free fatty acid (FA) uptake by interacting with several FA transport proteins. We previously reported that Cav3 knockout mice (Cav3KO) develop cardiac hypertrophy with diminished contractile function; however, the effects of Cav3 gene ablation on cardiac substrate utilization are unknown. The present study revealed that the uptake and oxidation of FAs and glucose were normal in hypertrophic Cav3KO hearts. Real-time PCR analysis revealed normal expression of lipid metabolism genes including FA translocase (CD36) and carnitine palmitoyl transferase-1 in Cav3KO hearts. Interestingly, myocardial cAMP content was significantly increased by 42%; however, this had no effect on PKA activity in Cav3KO hearts. Microarray expression analysis revealed a marked increase in the expression of genes involved in receptor trafficking to the plasma membrane, including Rab4a and the expression of WD repeat/FYVE domain containing proteins. We observed a fourfold increase in the expression of cellular retinol binding protein-III and a 3.5-fold increase in 17β-hydroxysteroid dehydrogenase type 11, a member of the short-chain dehydrogenase/reductase family involved in the biosynthesis and inactivation of steroid hormones. In summary, a loss of Cav3 in the heart leads to cardiac hypertrophy with normal substrate utilization. Moreover, a loss of Cav3 mRNA altered the expression of several genes not previously linked to cardiac growth and function. Thus we have identified a number of new target genes associated with the pathogenesis of cardiac hypertrophy. PMID:18552160

  12. 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

  13. Effects of trkB knockout on topography and ocular segregation of uncrossed retinal projections.

    PubMed

    Rodger, Jennifer; Frost, Douglas O

    2009-05-01

    TrkB is an important receptor for brain-derived neurotrophic factor and NT4, members of the neurotrophin family. TrkB signaling is crucial in many activity-dependent and activity-independent processes of neural development. Here, we investigate the role of trkB signaling in the development of two distinct, organizational features of retinal projections--the segregation of crossed and uncrossed retinal inputs along the "lines of projection" that represent a single point in the visual field and the "retinotopic" mapping of retinofugal axons within their cerebral targets. Using anterograde tracing, we obtained quantitative measures of the distribution of retinal projections in the dorsal nucleus of the lateral geniculate body (LGd) and superior colliculus (SC) of wild-type mice and mice homozygous for constitutive null mutation (knockout) of the full-length trkB receptor (trkB(FL)(-/-)). In trkB(FL)(-/-) mice, uncrossed retinal projections cluster normally but there is a topographic expansion in the distribution of these clusters across the SC. By contrast, the absence of trkB signaling has no significant effect on the segregation of crossed and uncrossed retinal projections along the lines of projection in LGd or SC. We conclude that the normal topographic organization of uncrossed retinal projections depends upon trkB signaling, whereas the segregation of crossed and uncrossed retinal projections is trkB-independent. We also found that in trkB(FL)(-/-) mice, neuronal number was reduced in the LGd and SC and in the caudate-putamen. Previous studies by ourselves and others have shown that the number of retinal ganglion cells (RGCs) is unchanged in trkB(FL)(-/-) mice. Together, these results demonstrate that there is no matching of the numbers of RGCs with neuronal numbers in the LGd or SC. PMID:19283373

  14. 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

  15. 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.

  16. 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.

  17. 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…

  18. 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

  19. Transcriptional control of behavior: Engrailed knockout changes cockroach escape trajectories

    PubMed Central

    Booth, David; Marie, Bruno; Domenici, Paolo; Blagburn, Jonathan M; Bacon, Jonathan P

    2009-01-01

    The cerci of the cockroach are covered with identified sensory hairs, which detect air movements. The sensory neurons which innervate these hairs synapse with giant interneurons (GIs) in the terminal ganglion which in turn synapse with interneurons and leg motorneurons in thoracic ganglia. This neural circuit mediates the animal's escape behavior. The transcription factor Engrailed (En) is expressed only in the medially born sensory neurons, which suggested it could work as a positional determinant of sensory neuron identity. Previously, we used dsRNA interference to abolish En expression, and found that the axonal arborization and synaptic outputs of an identified En-positive sensory neuron changed so that it came to resemble a nearby En-negative cell, which was itself unaffected. We thus demonstrated directly that En controls synaptic choice, as well as axon projections. Is escape behavior affected as a result of this mis-wiring? We recently showed that adult cockroaches keep each escape unpredictable by running along one of a set of preferred escape trajectories (ETs) at fixed angles from the direction of the threatening stimulus. The probability of selecting a particular ET is influenced by wind direction. In this present study we show that early instar juvenile cockroaches also use those same ETs. En knockout significantly perturbs the animals' perception of posterior wind, altering the choice of ETs to one more appropriate for anterior wind. This is the first time that it has been shown that knockout of a transcription factor controlling synaptic connectivity can alter the perception of a directional stimulus. PMID:19494140

  20. Bioelectric Characterization of Epithelia from Neonatal CFTR Knockout Ferrets

    PubMed Central

    Fisher, John T.; Tyler, Scott R.; Zhang, Yulong; Lee, Ben J.; Liu, Xiaoming; Sun, Xingshen; Sui, Hongshu; Liang, Bo; Luo, Meihui; Xie, Weiliang; Yi, Yaling; Zhou, Weihong; Song, Yi; Keiser, Nicholas; Wang, Kai; de Jonge, Hugo R.

    2013-01-01

    Cystic fibrosis (CF) is a life-shortening, recessive, multiorgan genetic disorder caused by the loss of CF transmembrane conductance regulator (CFTR) chloride channel function found in many types of epithelia. Animal models that recapitulate the human disease phenotype are critical to understanding pathophysiology in CF and developing therapies. CFTR knockout ferrets manifest many of the phenotypes observed in the human disease, including lung infections, pancreatic disease and diabetes, liver disease, malnutrition, and meconium ileus. In the present study, we have characterized abnormalities in the bioelectric properties of the trachea, stomach, intestine, and gallbladder of newborn CF ferrets. Short-circuit current (ISC) analysis of CF and wild-type (WT) tracheas revealed the following similarities and differences: (1) amiloride-sensitive sodium currents were similar between genotypes; (2) responses to 4,4′-diisothiocyano-2,2′-stilbene disulphonic acid were 3.3-fold greater in CF animals, suggesting elevated baseline chloride transport through non-CFTR channels in a subset of CF animals; and (3) a lack of 3-isobutyl-1-methylxanthine (IBMX)/forskolin–stimulated and N-(2-Naphthalenyl)-((3,5-dibromo-2,4-dihydroxyphenyl)methylene)glycine hydrazide (GlyH-101)–inhibited currents in CF animals due to the lack of CFTR. CFTR mRNA was present throughout all levels of the WT ferret and IBMX/forskolin–inducible ISC was only observed in WT animals. However, despite the lack of CFTR function in the knockout ferret, the luminal pH of the CF ferret gallbladder, stomach, and intestines was not significantly changed relative to WT. The WT stomach and gallbladder exhibited significantly enhanced IBMX/forskolin ISC responses and inhibition by GlyH-101 relative to CF samples. These findings demonstrate that multiple organs affected by disease in the CF ferret have bioelectric abnormalities consistent with the lack of cAMP-mediated chloride transport. PMID:23782101

  1. Preaxial Polydactyly in Sost/Sostdc1 Double Knockouts

    SciTech Connect

    Yee, C M; Collette, N M; Loots, G G

    2011-07-29

    In the United States, {approx}5% are born with congenital birth defects due to abnormal function of cellular processes and interactions. Sclerosteosis, a rare autosomal recessive disease, causes hyperostosis of the axial and appendicular skeleton, and patients present radial deviation, digit syndactyly, nail dysplasia, and overall high bone mineral density. Sclerosteosis is due to a loss of function of sclerostin (Sost). Sost is a Wnt (abbrev.) antagonist; when mutated, nonfunctional Sost results in hyperactive osteoblast activity which leads to abnormal high bone mass. Previous studies have shown that Sost overexpression in transgenic mice causes reduced bone mineral density and a variety of limb phenotypes ranging from lost, fused, and split phalanges. Consistent with clinical manifestations of Sclerosteosis, Sost knockout mice exhibit increased generalized bone mineral density and syndactyly of the digits. Sostdc1 is a paralog of Sost that has also been described as an antagonist of Wnt signaling, in developing tooth buds. Unlike Sost knockouts, Sostdc1 null mice do not display any limb abnormalities. To determine if Sost and Sostdc1 have redundant functions during limb patterning, we examined Sost; Sostdc1 mice determined that they exhibit a novel preaxial polydactyly phenotype with a low penetrance. LacZ staining, skeletal preparations, and in situ hybridization experiments were used to help characterize this novel phenotype and understand how this phenotype develops. We find Sost and Sostdc1 to have complementary expression patterns during limb development, and the loss of their expression alters the transcription of several key limb regulators, such as Fgf8, Shh and Grem.

  2. Estrogen Receptors, the Hippocampus, and Memory

    PubMed Central

    Bean, Linda A.; Ianov, Lara; Foster, Thomas C.

    2015-01-01

    Estradiol effects on memory depend on hormone levels and the interaction of different estrogen receptors within neural circuits. Estradiol induces gene transcription and rapid membrane signaling mediated by estrogen receptor-alpha (ERα), estrogen receptor-beta (ERβ), and a recently characterized G-protein coupled estrogen receptor, each with distinct distributions and ability to influence estradiol-dependent signaling. Investigations using receptor specific agonists suggest that all three receptors rapidly activate kinase-signaling and have complex dose-dependent influences on memory. Research employing receptor knockout mice demonstrate that ERα maintains transcription and memory as estradiol levels decline. This work indicates a regulatory role of ERβ in transcription and cognition, which depends on estradiol levels and the function of ERα. The regulatory role of ERβ is due in part to ERβ acting as a negative regulator of ERα-mediated transcription. Vector-mediated expression of estrogen receptors in the hippocampus provides an innovative research approach and suggests that memory depends on the relative expression of ERα and ERβ interacting with estradiol levels. Notably, the ability of estradiol to improve cognition declines with advanced age along with decreased expression of estrogen receptors. Thus, it will be important for future research to determine the mechanisms that regulate estrogen receptor expression during aging. PMID:24510074

  3. Health and population effects of rare gene knockouts in adult humans with related parents

    PubMed Central

    Narasimhan, Vagheesh M.; Hunt, Karen A.; Mason, Dan; Baker, Christopher L.; Karczewski, Konrad J.; Barnes, Michael R.; Barnett, Anthony H.; Bates, Chris; Bellary, Srikanth; Bockett, Nicholas A.; Giorda, Kristina; Griffiths, Christopher J.; Hemingway, Harry; Jia, Zhilong; Kelly, M. Ann; Khawaja, Hajrah A.; Lek, Monkol; McCarthy, Shane; McEachan, Rosie; O’Donnell-Luria, Anne; Paigen, Kenneth; Parisinos, Constantinos A.; Sheridan, Eamonn; Southgate, Laura; Tee, Louise; Thomas, Mark; Xue, Yali; Schnall-Levin, Michael; Petkov, Petko M.; Tyler-Smith, Chris; Maher, Eamonn R.; Trembath, Richard C.; MacArthur, Daniel G.; Wright, John; Durbin, Richard; van Heel, David A.

    2016-01-01

    Examining complete gene knockouts within a viable organism can inform on gene function. We sequenced the exomes of 3,222 British Pakistani-heritage adults with high parental relatedness, discovering 1,111 rare-variant homozygous genotypes with predicted loss of gene function (knockouts) in 781 genes. We observed 13.7% fewer than expected homozygous knockout genotypes, implying an average load of 1.6 recessive-lethal-equivalent LOF variants per adult. Linking genetic data to lifelong health records, knockouts were not associated with clinical consultation or prescription rate. In this dataset we identified a healthy PRDM9 knockout mother, and performed phased genome sequencing on her, her child and controls, which showed meiotic recombination sites localised away from PRDM9-dependent hotspots. Thus, natural LOF variants inform upon essential genetic loci, and demonstrate PRDM9 redundancy in humans. PMID:26940866

  4. Phenobarbital and Insulin Reciprocate Activation of the Nuclear Receptor Constitutive Androstane Receptor through the Insulin Receptor

    PubMed Central

    Yasujima, Tomoya; Saito, Kosuke; Moore, Rick

    2016-01-01

    Phenobarbital (PB) antagonized insulin to inactivate the insulin receptor and attenuated the insulin receptor downstream protein kinase B (AKT)–forkhead box protein O1 and extracellular signal-regulated kinase 1/2 signals in mouse primary hepatocytes and HepG2 cells. Hepatic AKT began dephosphorylation in an early stage of PB treatment, and blood glucose levels transiently increased in both wild-type and constitutive androstane receptor (CAR) knockout (KO) mice. On the other hand, blood glucose levels increased in wild-type mice, but not KO mice, in later stages of PB treatment. As a result, PB, acting as an insulin receptor antagonist, elicited CAR-independent increases and CAR-dependent decreases of blood glucose levels at these different stages of treatment, respectively. Reciprocally, insulin activation of the insulin receptor repressed CAR activation and induction of its target CYP2B6 gene in HepG2 cells. Thus, PB and insulin cross-talk through the insulin receptor to regulate glucose and drug metabolism reciprocally. PMID:26994072

  5. Phenobarbital and Insulin Reciprocate Activation of the Nuclear Receptor Constitutive Androstane Receptor through the Insulin Receptor.

    PubMed

    Yasujima, Tomoya; Saito, Kosuke; Moore, Rick; Negishi, Masahiko

    2016-05-01

    Phenobarbital (PB) antagonized insulin to inactivate the insulin receptor and attenuated the insulin receptor downstream protein kinase B (AKT)-forkhead box protein O1 and extracellular signal-regulated kinase 1/2 signals in mouse primary hepatocytes and HepG2 cells. Hepatic AKT began dephosphorylation in an early stage of PB treatment, and blood glucose levels transiently increased in both wild-type and constitutive androstane receptor (CAR) knockout (KO) mice. On the other hand, blood glucose levels increased in wild-type mice, but not KO mice, in later stages of PB treatment. As a result, PB, acting as an insulin receptor antagonist, elicited CAR-independent increases and CAR-dependent decreases of blood glucose levels at these different stages of treatment, respectively. Reciprocally, insulin activation of the insulin receptor repressed CAR activation and induction of its target CYP2B6 gene in HepG2 cells. Thus, PB and insulin cross-talk through the insulin receptor to regulate glucose and drug metabolism reciprocally.

  6. Roles of transferrin receptors in erythropoiesis.

    PubMed

    Kawabata, Hiroshi; Sakamoto, Soichiro; Masuda, Taro; Uchiyama, Tatsuki; Ohmori, Katsuyuki; Koeffler, H Phillip; Takaori-Kondo, Akifumi

    2016-07-01

    Erythropoiesis requires large amounts of iron for hemoglobin synthesis, which is mainly provided by macrophages and the intestines in a transferrin (Tf)-bound form. Bone marrow erythroblasts incorporate Tf through endocytosis, which is mediated by transferrin receptor 1 (TFR1). Recently, human TFR1, aside from its role as a Tf receptor, was also found to be a receptor for the H-subunit of ferritin (FTH). In humans, hematopoietic erythroid precursor cells express high levels of TFR1 and specifically take up the FTH homopolymer (H-ferritin). H-ferritin inhibits the formation of burst forming unit-erythroid colonies in vitro. TFR2, which is also a Tf receptor, is predominantly expressed in hepatocytes and erythroid precursor cells. In the liver, TFR2 forms a complex with HFE, a hereditary hemochromatosis-associated protein, and acts as an iron sensor. In mice, hepatocyte-specific knockout of the TFR2 gene has been shown to cause systemic iron-overload with decreased expression of hepcidin, the central regulator of iron homeostasis. In erythroid cells, TFR2 forms a complex with the erythropoietin receptor and facilitates its trafficking to the cell membrane. Moreover, hematopoietic cell-specific knockout of the TFR2 gene causes microcytic erythrocytosis in mice. This review focuses on the molecular evolution and functions of these TFRs and their ligands. PMID:27498743

  7. Lipoprotein Receptors Redundantly Participate in Entry of Hepatitis C Virus

    PubMed Central

    Ono, Chikako; Uemura, Kentaro; Kawachi, Yukako; Shiokawa, Mai; Mori, Hiroyuki; Wada, Masami; Shima, Ryoichi; Okamoto, Toru; Hiraga, Nobuhiko; Suzuki, Ryosuke; Chayama, Kazuaki; Wakita, Takaji; Matsuura, Yoshiharu

    2016-01-01

    Scavenger receptor class B type 1 (SR-B1) and low-density lipoprotein receptor (LDLR) are known to be involved in entry of hepatitis C virus (HCV), but their precise roles and their interplay are not fully understood. In this study, deficiency of both SR-B1 and LDLR in Huh7 cells was shown to impair the entry of HCV more strongly than deficiency of either SR-B1 or LDLR alone. In addition, exogenous expression of not only SR-B1 and LDLR but also very low-density lipoprotein receptor (VLDLR) rescued HCV entry in the SR-B1 and LDLR double-knockout cells, suggesting that VLDLR has similar roles in HCV entry. VLDLR is a lipoprotein receptor, but the level of its hepatic expression was lower than those of SR-B1 and LDLR. Moreover, expression of mutant lipoprotein receptors incapable of binding to or uptake of lipid resulted in no or slight enhancement of HCV entry in the double-knockout cells, suggesting that binding and/or uptake activities of lipid by lipoprotein receptors are essential for HCV entry. In addition, rescue of infectivity in the double-knockout cells by the expression of the lipoprotein receptors was not observed following infection with pseudotype particles bearing HCV envelope proteins produced in non-hepatic cells, suggesting that lipoproteins associated with HCV particles participate in the entry through their interaction with lipoprotein receptors. Buoyant density gradient analysis revealed that HCV utilizes these lipoprotein receptors in a manner dependent on the lipoproteins associated with HCV particles. Collectively, these results suggest that lipoprotein receptors redundantly participate in the entry of HCV. PMID:27152966

  8. Enhanced response of T lymphocytes from Pgap3 knockout mouse: Insight into roles of fatty acid remodeling of GPI anchored proteins.

    PubMed

    Murakami, Hidekazu; Wang, Yetao; Hasuwa, Hidetoshi; Maeda, Yusuke; Kinoshita, Taroh; Murakami, Yoshiko

    2012-01-27

    Glycosylphosphatidylinositol (GPI) is a complex glycolipid that serves as a membrane anchor for many cell-surface proteins, such as Thy-1 and CD48. GPI-anchored proteins (GPI-APs) play important roles in many biological processes, such as signal transduction and cell-cell interaction, through their association with lipid rafts. Fatty acid remodeling of GPI-APs in the Golgi apparatus is required for their efficient association with lipid rafts, i.e., the unsaturated fatty acid at the sn-2 position of the PI moiety is exchanged for the saturated fatty acid by PGAP2 and PGAP3. To investigate the immunological role of the fatty acid remodeling of GPI-APs, we generated a Pgap3 knockout mouse. In this mouse, GPI-APs are expressed on the cell surface without fatty acid remodeling, and fail to associate with lipid rafts. Male Pgap3 knockout mice were born alive at a ratio lower than expected from Mendel's law, whereas the number of female mice followed Mendel's law. All mice exhibited growth retardation and abnormal reflexes such as limb grasping. We focused T cell function in these mice and found that T cell development in the absence of Pgap3 was normal. However, the response of T cells was enhanced in Pgap3 knockout mice in both in vitro and in vivo studies, including alloreactive response, antigen-specific immune response, and experimental autoimmune encephalomyelitis. Cross-linking of Thy-1 in wild-type cells inhibited the signal transduced by the T cell receptor (TCR), whereas cross-linking of Thy-1 in Pgap3 knockout cells enhanced the TCR signal. These results suggest that GPI-APs localized in lipid rafts may modulate signaling through the TCR.

  9. Endothelial glucocorticoid receptor suppresses atherogenesis- Brief Report

    PubMed Central

    Zhang, Xinbo; Rotllan, Noemi; Feng, Yan; Zhou, Han; Fernández-Hernando, Carlos; Yu, Jun; Sessa, William C.

    2015-01-01

    Objective The purpose of this study was to determine the role of the endothelial glucocorticoid receptor in the pathogenesis of atherosclerosis. Approach and Results Control mice and mice lacking the endothelial glucocorticoid receptor were bred onto an Apoe knockout background and subjected to high-fat diet feeding for 12 weeks. Assessment of body weight and total cholesterol and triglycerides before and after the diet revealed no differences between the two groups of mice. However, mice lacking the endothelial glucocorticoid receptor developed more severe atherosclerotic lesions in the aorta, brachiocephalic artery and aortic sinus as well as a heightened inflammatory milieu as evidence by increased macrophage recruitment in the lesions. Conclusions These data suggest the endothelial glucocorticoid receptor is important for tonic inhibition of inflammation and limitation of atherosclerosis progression in this model. PMID:25810297

  10. Estrogen receptor signaling during vertebrate development

    PubMed Central

    Bondesson, Maria; Hao, Ruixin; Lin, Chin-Yo; Williams, Cecilia; Gustafsson, Jan-Åke

    2014-01-01

    Estrogen receptors are expressed and their cognate ligands produced in all vertebrates, indicative of important and conserved functions. Through evolution estrogen has been involved in controlling reproduction, affecting both the development of reproductive organs and reproductive behavior. This review broadly describes the synthesis of estrogens and the expression patterns of aromatase and the estrogen receptors, in relation to estrogen functions in the developing fetus and child. We focus on the role of estrogens for development of reproductive tissues, as well as non-reproductive effects on the developing brain. We collate data from human, rodent, bird and fish studies and highlight common and species-specific effects of estrogen signaling on fetal development. Morphological malformations originating from perturbed estrogen signaling in estrogen receptor and aromatase knockout mice are discussed, as well as the clinical manifestations of rare estrogen receptor alpha and aromatase gene mutations in humans. PMID:24954179

  11. Genetic approaches to unraveling G protein-coupled receptor biology.

    PubMed

    Aparicio, Samuel A J R; Powell, Justin

    2004-09-01

    Genetic approaches to validating G protein-coupled receptors (GPCRs) have proven to be a powerful research tool, especially knockout studies in rodents. To date, data related to in vivo function have been published on approximately half of the human rhodopsin-like family-1 GPCRs, which can be attributed to the use of mouse knockouts. It is likely that many currently unknown yet important therapeutic mechanisms will be uncovered through knockout screens in mice. One such recent discovery is the elucidation of the in vivo function of the GPCR GPR54 through mouse genetics, and its subsequent validation in human populations. Although previously unsuspected, GPR54 has been found to be a master-regulator of the hypothalamic-pituitary-gonadal axis.

  12. [An efficient genetic knockout system based on linear DNA fragment homologous recombination for halophilic archaea].

    PubMed

    Xiaoli, Wang; Chuang, Jiang; Jianhua, Liu; Xipeng, Liu

    2015-04-01

    With the development of functional genomics, gene-knockout is becoming an important tool to elucidate gene functions in vivo. As a good model strain for archaeal genetics, Haloferax volcanii has received more attention. Although several genetic manipulation systems have been developed for some halophilic archaea, it is time-consuming because of the low percentage of positive clones during the second-recombination selection. These classical gene knockout methods are based on DNA recombination between the genomic homologous sequence and the circular suicide plasmid, which carries a pyrE selection marker and two DNA fragments homologous to the upstream and downstream fragments of the target gene. Many wild-type clones are obtained through a reverse recombination between the plasmid and genome in the classic gene knockout method. Therefore, it is necessary to develop an efficient gene knockout system to increase the positive clone percentage. Here we report an improved gene knockout method using a linear DNA cassette consisting of upstream and downstream homologous fragments, and the pyrE marker. Gene deletions were subsequently detected by colony PCR analysis. We determined the efficiency of our knockout method by deleting the xpb2 gene from the H. volcanii genome, with the percentage of positive clones higher than 50%. Our method provides an efficient gene knockout strategy for halophilic archaea.

  13. Final-state interactions in two-nucleon knockout reactions

    NASA Astrophysics Data System (ADS)

    Colle, Camille; Cosyn, Wim; Ryckebusch, Jan

    2016-03-01

    Background: Exclusive two-nucleon knockout after electroexcitation of nuclei [A (e ,e'N N ) in brief] is considered to be a primary source of information about short-range correlations (SRCs) in nuclei. For a proper interpretation of the data, final-state interactions (FSIs) need to be theoretically controlled. Purpose: Our goal is to quantify the role of FSI effects in exclusive A (e ,e'p N ) reactions for four target nuclei representative of the whole mass region. Our focus is on processes that are SRC driven. We investigate the role of FSIs for two characteristic detector setups corresponding to "small" and "large" coverage of the available phase space. Method: Use is made of a factorized expression for the A (e ,e'p N ) cross section that is proportional to the two-body center-of-mass (c.m.) momentum distribution of close-proximity pairs. The A (e ,e'p p ) and A (e ,e'p n ) reactions for the target nuclei 12C,27Al,56Fe, and 208Pb are investigated. The elastic attenuation mechanisms in the FSIs are included using the relativistic multiple-scattering Glauber approximation (RMSGA). Single-charge exchange (SCX) reactions are also included. We introduce the nuclear transparency TAp N, defined as the ratio of exclusive (e ,e'p N ) cross sections on nuclei to those on "free" nucleon pairs, as a measure for the aggregated effect of FSIs in p N knockout reactions from nucleus A . A toy model is introduced in order to gain a better understanding of the A dependence of TAp N. Results: The transparency TAp N drops from 0.2 -0.3 for 12C to 0.04 -0.07 for 208Pb. For all considered kinematics, the mass dependence of TAp N can be captured by the power law TAp N∝A-λ with 0.4 ≲λ ≲0.5 . Apart from an overall reduction factor, we find that FSIs only modestly affect the distinct features of SRC-driven A (e ,e'p N ) which are dictated by the c.m. distribution of close-proximity pairs. Conclusion: The SCX mechanisms represent a relatively small (order of a few percent

  14. Mouse ataxin-3 functional knock-out model.

    PubMed

    Switonski, Pawel M; Fiszer, Agnieszka; Kazmierska, Katarzyna; Kurpisz, Maciej; Krzyzosiak, Wlodzimierz J; Figiel, Maciej

    2011-03-01

    Spinocerebellar ataxia 3 (SCA3) is a genetic disorder resulting from the expansion of the CAG repeats in the ATXN3 gene. The pathogenesis of SCA3 is based on the toxic function of the mutant ataxin-3 protein, but the exact mechanism of the disease remains elusive. Various types of transgenic mouse models explore different aspects of SCA3 pathogenesis, but a knock-in humanized mouse has not yet been created. The initial aim of this study was to generate an ataxin-3 humanized mouse model using a knock-in strategy. The human cDNA for ataxin-3 containing 69 CAG repeats was cloned from SCA3 patient and introduced into the mouse ataxin-3 locus at exon 2, deleting it along with exon 3 and intron 2. Although the human transgene was inserted correctly, the resulting mice acquired the knock-out properties and did not express ataxin-3 protein in any analyzed tissues, as confirmed by western blot and immunohistochemistry. Analyses of RNA expression revealed that the entire locus consisting of human and mouse exons was expressed and alternatively spliced. We detected mRNA isoforms composed of exon 1 spliced with mouse exon 4 or with human exon 7. After applying 37 PCR cycles, we also detected a very low level of the correct exon 1/exon 2 isoform. Additionally, we confirmed by bioinformatic analysis that the structure and power of the splicing site between mouse intron 1 and human exon 2 (the targeted locus) was not changed compared with the native mouse locus. We hypothesized that these splicing aberrations result from the deletion of further splicing sites and the presence of a strong splicing site in exon 4, which was confirmed by bioinformatic analysis. In summary, we created a functional ataxin-3 knock-out mouse model that is viable and fertile and does not present a reduced life span. Our work provides new insights into the splicing characteristics of the Atxn3 gene and provides useful information for future attempts to create knock-in SCA3 models.

  15. The hallucinogen N,N-dimethyltryptamine (DMT) is an endogenous sigma-1 receptor regulator.

    PubMed

    Fontanilla, Dominique; Johannessen, Molly; Hajipour, Abdol R; Cozzi, Nicholas V; Jackson, Meyer B; Ruoho, Arnold E

    2009-02-13

    The sigma-1 receptor is widely distributed in the central nervous system and periphery. Originally mischaracterized as an opioid receptor, the sigma-1 receptor binds a vast number of synthetic compounds but does not bind opioid peptides; it is currently considered an orphan receptor. The sigma-1 receptor pharmacophore includes an alkylamine core, also found in the endogenous compound N,N-dimethyltryptamine (DMT). DMT acts as a hallucinogen, but its receptor target has been unclear. DMT bound to sigma-1 receptors and inhibited voltage-gated sodium ion (Na+) channels in both native cardiac myocytes and heterologous cells that express sigma-1 receptors. DMT induced hypermobility in wild-type mice but not in sigma-1 receptor knockout mice. These biochemical, physiological, and behavioral experiments indicate that DMT is an endogenous agonist for the sigma-1 receptor.

  16. Modeling fragile X syndrome in the Fmr1 knockout mouse

    PubMed Central

    Kazdoba, Tatiana M.; Leach, Prescott T.; Silverman, Jill L.; Crawley, Jacqueline N.

    2014-01-01

    Summary Fragile X Syndrome (FXS) is a commonly inherited form of intellectual disability and one of the leading genetic causes for autism spectrum disorder. Clinical symptoms of FXS can include impaired cognition, anxiety, hyperactivity, social phobia, and repetitive behaviors. FXS is caused by a CGG repeat mutation which expands a region on the X chromosome containing the FMR1 gene. In FXS, a full mutation (> 200 repeats) leads to hypermethylation of FMR1, an epigenetic mechanism that effectively silences FMR1 gene expression and reduces levels of the FMR1 gene product, fragile X mental retardation protein (FMRP). FMRP is an RNA-binding protein that is important for the regulation of protein expression. In an effort to further understand how loss of FMR1 and FMRP contribute to FXS symptomology, several FXS animal models have been created. The most well characterized rodent model is the Fmr1 knockout (KO) mouse, which lacks FMRP protein due to a disruption in its Fmr1 gene. Here, we review the behavioral phenotyping of the Fmr1 KO mouse to date, and discuss the clinical relevance of this mouse model to the human FXS condition. While much remains to be learned about FXS, the Fmr1 KO mouse is a valuable tool for understanding the repercussions of functional loss of FMRP and assessing the efficacy of pharmacological compounds in ameliorating the molecular and behavioral phenotypes relevant to FXS. PMID:25606362

  17. 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

  18. NELF knockout is associated with impaired pubertal development and subfertility.

    PubMed

    Quaynor, Samuel D; Ko, Eun Kyung; Chorich, Lynn P; Sullivan, Megan E; Demir, Durkadin; Waller, Jennifer L; Kim, Hyung-Goo; Cameron, Richard S; Layman, Lawrence C

    2015-05-15

    Puberty and reproduction require proper signaling of the hypothalamic-pituitary-gonadal axis controlled by gonadotropin-releasing hormone (GnRH) neurons, which arise in the olfactory placode region and migrate along olfactory axons to the hypothalamus. Factors adversely affecting GnRH neuron specification, migration, and function lead to delayed puberty and infertility. Nasal embryonic luteinizing hormone-releasing factor (NELF) is a predominantly nuclear protein. NELF mutations have been demonstrated in patients with hypogonadotropic hypogonadism, but biallelic mutations are rare and heterozygous NELF mutations typically co-exist with mutations in another gene. Our previous studies in immortalized GnRH neurons supported a role for NELF in GnRH neuron migration. To better understand the physiology of NELF, a homozygous Nelf knockout (KO) mouse model was generated. Our findings indicate that female Nelf KO mice have delayed vaginal opening but no delay in time to first estrus, decreased uterine weight, and reduced GnRH neuron number. In contrast, male mice were normal at puberty. Both sexes of mice had impaired fertility manifested as reduced mean litter size. These data support that NELF has important reproductive functions. The milder than expected phenotype of KO mice also recapitulates the human phenotype since heterozygous NELF mutations usually require an additional mutation in a second gene to result in hypogonadotropic hypogonadism.

  19. Gastrointestinal Pathology in Juvenile and Adult CFTR-Knockout Ferrets

    PubMed Central

    Sun, Xingshen; Olivier, Alicia K.; Yi, Yaling; Pope, Christopher E.; Hayden, Hillary S.; Liang, Bo; Sui, Hongshu; Zhou, Weihong; Hager, Kyle R.; Zhang, Yulong; Liu, Xiaoming; Yan, Ziying; Fisher, John T.; Keiser, Nicholas W.; Song, Yi; Tyler, Scott R.; Goeken, J. Adam; Kinyon, Joann M.; Radey, Matthew C.; Fligg, Danielle; Wang, Xiaoyan; Xie, Weiliang; Lynch, Thomas J.; Kaminsky, Paul M.; Brittnacher, Mitchell J.; Miller, Samuel I.; Parekh, Kalpaj; Meyerholz, David K.; Hoffman, Lucas R.; Frana, Timothy; Stewart, Zoe A.; Engelhardt, John F.

    2015-01-01

    Cystic fibrosis (CF) is a multiorgan disease caused by loss of a functional cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel in many epithelia of the body. Here we report the pathology observed in the gastrointestinal organs of juvenile to adult CFTR-knockout ferrets. CF gastrointestinal manifestations included gastric ulceration, intestinal bacterial overgrowth with villous atrophy, and rectal prolapse. Metagenomic phylogenetic analysis of fecal microbiota by deep sequencing revealed considerable genotype-independent microbial diversity between animals, with the majority of taxa overlapping between CF and non-CF pairs. CF hepatic manifestations were variable, but included steatosis, necrosis, biliary hyperplasia, and biliary fibrosis. Gallbladder cystic mucosal hyperplasia was commonly found in 67% of CF animals. The majority of CF animals (85%) had pancreatic abnormalities, including extensive fibrosis, loss of exocrine pancreas, and islet disorganization. Interestingly, 2 of 13 CF animals retained predominantly normal pancreatic histology (84% to 94%) at time of death. Fecal elastase-1 levels from these CF animals were similar to non-CF controls, whereas all other CF animals evaluated were pancreatic insufficient (<2 μg elastase-1 per gram of feces). These findings suggest that genetic factors likely influence the extent of exocrine pancreas disease in CF ferrets and have implications for the etiology of pancreatic sufficiency in CF patients. In summary, these studies demonstrate that the CF ferret model develops gastrointestinal pathology similar to CF patients. PMID:24637292

  20. Adaptation of the myoglobin knockout mouse to hypoxic stress.

    PubMed

    Schlieper, Georg; Kim, Jie-Hoon; Molojavyi, Andrei; Jacoby, Christoph; Laussmann, Tim; Flögel, Ulrich; Gödecke, Axel; Schrader, Jürgen

    2004-04-01

    Myoglobin knockout (myo-/-) mice were previously reported to show no obvious phenotype but revealed several compensatory mechanisms that include increases in cardiac capillary density, coronary flow, and hemoglobin. The aim of this study was to investigate whether severe hypoxic stress can exhaust these compensatory mechanisms and whether this can be monitored on the gene and protein level. Myo-/- and wild-type (WT) mice we e exposed to hypoxia (10% O(2)) fo 2 wk. Thereafter hemodynamic parameters were investigated by invasive measurement combined with magnetic resonance imaging. Cardiac gene and protein expression were analyzed using cDNA arrays and two-dimensional gel electrophoresis plus mass spectrometry, respectively. Hematocrit levels increased from 44% (WT) and 48% (myo-/-) to 72% in both groups. Similar to WT controls, hypoxic myo-/- animals maintained stable cardiovascular function (mean arterial blood pressure 82.4 mmHg, ejection fraction 72.5%). Cardiac gene expression of hypoxic myo-/- mice differed significantly from WT controls in 17 genes (e.g., keratinocyte lipid binding protein +202%, cytochrome c oxidase Vb +41%). Interestingly, hypoxia inducible factor-1alpha remained unchanged in both groups. Proteome analysis revealed reduced levels of heart fatty acid-binding protein and heat shock protein 27 both in hypoxic myo-/- and WT mice. Our data thus demonstrate that myo-/- mice do not decompensate du ing hypoxic st ess but a e surprisingly well adapted. Changes in ene gy metabolism of fatty acids may contribute to the robustness of myoglobin-deficient mice. PMID:14656764

  1. 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

  2. The biology of novel animal genes: Mouse APEX gene knockout

    SciTech Connect

    MacInnes, M.; Altherr, M.R.; Ludwig, D.; Pedersen, R.; Mold, C.

    1997-07-01

    This is the final report of a one-year, Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). The controlled breeding of novel genes into mice, including the gene knockout (KO), or conversely by adding back transgenes provide powerful genetic technologies that together suffice to determine in large part the biological role(s) of novel genes. Inbred mouse remains the best understood and most useful mammalian experimental system available for tackling the biology of novel genes. The major mammalian apurinic/apyrimidinic (AP) endonuclease (APE), is involved in a key step in the repair of spontaneous and induced AP sites in DNA. Efficient repair of these lesions is imperative to prevent the stable incorporation of mutations into the cellular genome which may lead to cell death or transformation. Loss or modulation of base excison repair activity in vivo may elevate the spontaneous mutation rate in cells, and may lead to a substantial increase in the incidence of cancer. Despite extensive biochemical analysis, however, the significance of these individual APE functions in vivo has not been elucidated. Mouse embryonic stem (ES) cells heterozygous for a deletion mutation in APE have been generated and whole animals containing the APE mutation have been derived from these ES cells. Animals homozygous for the APE null mutation die early in gestation, underscoring the biological significance of this DNA repair gene.

  3. Adaptation of the myoglobin knockout mouse to hypoxic stress.

    PubMed

    Schlieper, Georg; Kim, Jie-Hoon; Molojavyi, Andrei; Jacoby, Christoph; Laussmann, Tim; Flögel, Ulrich; Gödecke, Axel; Schrader, Jürgen

    2004-04-01

    Myoglobin knockout (myo-/-) mice were previously reported to show no obvious phenotype but revealed several compensatory mechanisms that include increases in cardiac capillary density, coronary flow, and hemoglobin. The aim of this study was to investigate whether severe hypoxic stress can exhaust these compensatory mechanisms and whether this can be monitored on the gene and protein level. Myo-/- and wild-type (WT) mice we e exposed to hypoxia (10% O(2)) fo 2 wk. Thereafter hemodynamic parameters were investigated by invasive measurement combined with magnetic resonance imaging. Cardiac gene and protein expression were analyzed using cDNA arrays and two-dimensional gel electrophoresis plus mass spectrometry, respectively. Hematocrit levels increased from 44% (WT) and 48% (myo-/-) to 72% in both groups. Similar to WT controls, hypoxic myo-/- animals maintained stable cardiovascular function (mean arterial blood pressure 82.4 mmHg, ejection fraction 72.5%). Cardiac gene expression of hypoxic myo-/- mice differed significantly from WT controls in 17 genes (e.g., keratinocyte lipid binding protein +202%, cytochrome c oxidase Vb +41%). Interestingly, hypoxia inducible factor-1alpha remained unchanged in both groups. Proteome analysis revealed reduced levels of heart fatty acid-binding protein and heat shock protein 27 both in hypoxic myo-/- and WT mice. Our data thus demonstrate that myo-/- mice do not decompensate du ing hypoxic st ess but a e surprisingly well adapted. Changes in ene gy metabolism of fatty acids may contribute to the robustness of myoglobin-deficient mice.

  4. The truncated TrkB receptor influences mammalian sleep

    PubMed Central

    Watson, Adam J.; Henson, Kyle; Dorsey, Susan G.

    2014-01-01

    Brain-derived neurotrophic factor (BDNF) is a neurotrophin hypothesized to play an important role in mammalian sleep expression and regulation. In order to investigate the role of the truncated receptor for BDNF, TrkB.T1, in mammalian sleep, we examined sleep architecture and sleep regulation in adult mice constitutively lacking this receptor. We find that TrkB.T1 knockout mice have increased REM sleep time, reduced REM sleep latency, and reduced sleep continuity. These results demonstrate a novel role for the TrkB.T1 receptor in sleep expression and provide new insights into the relationship between BDNF, psychiatric illness, and sleep. PMID:25502751

  5. Insights from the Study of Animals Lacking Functional Estrogen Receptor

    NASA Astrophysics Data System (ADS)

    Korach, Kenneth S.

    1994-12-01

    Estrogen hormones produce physiological actions within a variety of target sites in the body and during development by activating a specific receptor protein. Hormone responsiveness for the estrogen receptor protein was investigated at different stages of development with the use of gene knockout techniques because no natural genetic mutants have been described. A mutant mouse line without a functional estrogen receptor was created and is being used to assess estrogen responsiveness. Both sexes of these mutant animals are infertile and show a variety of phenotypic changes, some of which are associated with the gonads, mammary glands, reproductive tracts, and skeletal tissues.

  6. Sweet taste receptor deficient mice have decreased adiposity and increased bone mass.

    PubMed

    Simon, Becky R; Learman, Brian S; Parlee, Sebastian D; Scheller, Erica L; Mori, Hiroyuki; Cawthorn, William P; Ning, Xiaomin; Krishnan, Venkatesh; Ma, Yanfei L; Tyrberg, Björn; MacDougald, Ormond A

    2014-01-01

    Functional expression of sweet taste receptors (T1R2 and T1R3) has been reported in numerous metabolic tissues, including the gut, pancreas, and, more recently, in adipose tissue. It has been suggested that sweet taste receptors in these non-gustatory tissues may play a role in systemic energy balance and metabolism. Smaller adipose depots have been reported in T1R3 knockout mice on a high carbohydrate diet, and sweet taste receptors have been reported to regulate adipogenesis in vitro. To assess the potential contribution of sweet taste receptors to adipose tissue biology, we investigated the adipose tissue phenotypes of T1R2 and T1R3 knockout mice. Here we provide data to demonstrate that when fed an obesogenic diet, both T1R2 and T1R3 knockout mice have reduced adiposity and smaller adipocytes. Although a mild glucose intolerance was observed with T1R3 deficiency, other metabolic variables analyzed were similar between genotypes. In addition, food intake, respiratory quotient, oxygen consumption, and physical activity were unchanged in T1R2 knockout mice. Although T1R2 deficiency did not affect adipocyte number in peripheral adipose depots, the number of bone marrow adipocytes is significantly reduced in these knockout animals. Finally, we present data demonstrating that T1R2 and T1R3 knockout mice have increased cortical bone mass and trabecular remodeling. This report identifies novel functions for sweet taste receptors in the regulation of adipose and bone biology, and suggests that in these contexts, T1R2 and T1R3 are either dependent on each other for activity or have common independent effects in vivo. PMID:24466105

  7. 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.

  8. 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

  9. 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

  10. 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

  11. 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.

  12. 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

  13. 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

  14. Thermoregulatory phenotype of the Trpv1 knockout mouse: thermoeffector dysbalance with hyperkinesis.

    PubMed

    Garami, Andras; Pakai, Eszter; Oliveira, Daniela L; Steiner, Alexandre A; Wanner, Samuel P; Almeida, M Camila; Lesnikov, Vladimir A; Gavva, Narender R; Romanovsky, Andrej A

    2011-02-01

    This study aimed at determining the thermoregulatory phenotype of mice lacking transient receptor potential vanilloid-1 (TRPV1) channels. We used Trpv1 knockout (KO) mice and their genetically unaltered littermates to study diurnal variations in deep body temperature (T(b)) and thermoeffector activities under basal conditions, as well as thermoregulatory responses to severe heat and cold. Only subtle alterations were found in the basal T(b) of Trpv1 KO mice or in their T(b) responses to thermal challenges. The main thermoregulatory abnormality of Trpv1 KO mice was a different pattern of thermoeffectors used to regulate T(b). On the autonomic side, Trpv1 KO mice were hypometabolic (had a lower oxygen consumption) and hypervasoconstricted (had a lower tail skin temperature). In agreement with the enhanced skin vasoconstriction, Trpv1 KO mice had a higher thermoneutral zone. On the behavioral side, Trpv1 KO mice preferred a lower ambient temperature and expressed a higher locomotor activity. Experiments with pharmacological TRPV1 agonists (resiniferatoxin and anandamide) and a TRPV1 antagonist (AMG0347) confirmed that TRPV1 channels located outside the brain tonically inhibit locomotor activity. With age (observed for up to 14 months), the body mass of Trpv1 KO mice exceeded that of controls, sometimes approaching 60 g. In summary, Trpv1 KO mice possess a distinct thermoregulatory phenotype, which is coupled with a predisposition to age-associated overweight and includes hypometabolism, enhanced skin vasoconstriction, decreased thermopreferendum, and hyperkinesis. The latter may be one of the primary deficiencies in Trpv1 KO mice. We propose that TRPV1-mediated signals from the periphery tonically suppress the general locomotor activity.

  15. A Jacob/Nsmf Gene Knockout Results in Hippocampal Dysplasia and Impaired BDNF Signaling in Dendritogenesis

    PubMed Central

    Schumacher, Anne; Butnaru, Ioana; Macharadze, Tamar; Gomes, Guilherme M.; Yuanxiang, PingAn; Bayraktar, Gonca; Rodenstein, Carolin; Geiseler, Carolin; Kolodziej, Angela; Lopez-Rojas, Jeffrey; Montag, Dirk; Angenstein, Frank; Bär, Julia; D’Hanis, Wolfgang; Roskoden, Thomas; Mikhaylova, Marina; Budinger, Eike; Ohl, Frank W.; Stork, Oliver; Zenclussen, Ana C.; Karpova, Anna; Schwegler, Herbert; Kreutz, Michael R.

    2016-01-01

    Jacob, the protein encoded by the Nsmf gene, is involved in synapto-nuclear signaling and docks an N-Methyl-D-Aspartate receptor (NMDAR)-derived signalosome to nuclear target sites like the transcription factor cAMP-response-element-binding protein (CREB). Several reports indicate that mutations in NSMF are related to Kallmann syndrome (KS), a neurodevelopmental disorder characterized by idiopathic hypogonadotropic hypogonadism (IHH) associated with anosmia or hyposmia. It has also been reported that a protein knockdown results in migration deficits of Gonadotropin-releasing hormone (GnRH) positive neurons from the olfactory bulb to the hypothalamus during early neuronal development. Here we show that mice that are constitutively deficient for the Nsmf gene do not present phenotypic characteristics related to KS. Instead, these mice exhibit hippoc