WorldWideScience

Sample records for receptor subunits contribute

  1. GABAA Receptors Containing ρ1 Subunits Contribute to In Vivo Effects of Ethanol in Mice

    Science.gov (United States)

    Blednov, Yuri A.; Benavidez, Jillian M.; Black, Mendy; Leiter, Courtney R.; Osterndorff-Kahanek, Elizabeth; Johnson, David; Borghese, Cecilia M.; Hanrahan, Jane R.; Johnston, Graham A. R.; Chebib, Mary; Harris, R. Adron

    2014-01-01

    GABAA receptors consisting of ρ1, ρ2, or ρ3 subunits in homo- or hetero-pentamers have been studied mainly in retina but are detected in many brain regions. Receptors formed from ρ1 are inhibited by low ethanol concentrations, and family-based association analyses have linked ρ subunit genes with alcohol dependence. We determined if genetic deletion of ρ1 in mice altered in vivo ethanol effects. Null mutant male mice showed reduced ethanol consumption and preference in a two-bottle choice test with no differences in preference for saccharin or quinine. Null mutant mice of both sexes demonstrated longer duration of ethanol-induced loss of righting reflex (LORR), and males were more sensitive to ethanol-induced motor sedation. In contrast, ρ1 null mice showed faster recovery from acute motor incoordination produced by ethanol. Null mutant females were less sensitive to ethanol-induced development of conditioned taste aversion. Measurement of mRNA levels in cerebellum showed that deletion of ρ1 did not change expression of ρ2, α2, or α6 GABAA receptor subunits. (S)-4-amino-cyclopent-1-enyl butylphosphinic acid (“ρ1” antagonist), when administered to wild type mice, mimicked the changes that ethanol induced in ρ1 null mice (LORR and rotarod tests), but the ρ1 antagonist did not produce these effects in ρ1 null mice. In contrast, (R)-4-amino-cyclopent-1-enyl butylphosphinic acid (“ρ2” antagonist) did not change ethanol actions in wild type but produced effects in mice lacking ρ1 that were opposite of the effects of deleting (or inhibiting) ρ1. These results suggest that ρ1 has a predominant role in two in vivo effects of ethanol, and a role for ρ2 may be revealed when ρ1 is deleted. We also found that ethanol produces similar inhibition of function of recombinant ρ1 and ρ2 receptors. These data indicate that ethanol action on GABAA receptors containing ρ1/ρ2 subunits may be important for specific effects of ethanol in vivo. PMID:24454882

  2. GABAA receptors containing ρ1 subunits contribute to in vivo effects of ethanol in mice.

    Directory of Open Access Journals (Sweden)

    Yuri A Blednov

    Full Text Available GABAA receptors consisting of ρ1, ρ2, or ρ3 subunits in homo- or hetero-pentamers have been studied mainly in retina but are detected in many brain regions. Receptors formed from ρ1 are inhibited by low ethanol concentrations, and family-based association analyses have linked ρ subunit genes with alcohol dependence. We determined if genetic deletion of ρ1 in mice altered in vivo ethanol effects. Null mutant male mice showed reduced ethanol consumption and preference in a two-bottle choice test with no differences in preference for saccharin or quinine. Null mutant mice of both sexes demonstrated longer duration of ethanol-induced loss of righting reflex (LORR, and males were more sensitive to ethanol-induced motor sedation. In contrast, ρ1 null mice showed faster recovery from acute motor incoordination produced by ethanol. Null mutant females were less sensitive to ethanol-induced development of conditioned taste aversion. Measurement of mRNA levels in cerebellum showed that deletion of ρ1 did not change expression of ρ2, α2, or α6 GABAA receptor subunits. (S-4-amino-cyclopent-1-enyl butylphosphinic acid ("ρ1" antagonist, when administered to wild type mice, mimicked the changes that ethanol induced in ρ1 null mice (LORR and rotarod tests, but the ρ1 antagonist did not produce these effects in ρ1 null mice. In contrast, (R-4-amino-cyclopent-1-enyl butylphosphinic acid ("ρ2" antagonist did not change ethanol actions in wild type but produced effects in mice lacking ρ1 that were opposite of the effects of deleting (or inhibiting ρ1. These results suggest that ρ1 has a predominant role in two in vivo effects of ethanol, and a role for ρ2 may be revealed when ρ1 is deleted. We also found that ethanol produces similar inhibition of function of recombinant ρ1 and ρ2 receptors. These data indicate that ethanol action on GABAA receptors containing ρ1/ρ2 subunits may be important for specific effects of ethanol in vivo.

  3. Decreased surface expression of the δ subunit of the GABAA receptor contributes to reduced tonic inhibition in dentate granule cells in a mouse model of fragile X syndrome.

    Science.gov (United States)

    Zhang, Nianhui; Peng, Zechun; Tong, Xiaoping; Lindemeyer, A Kerstin; Cetina, Yliana; Huang, Christine S; Olsen, Richard W; Otis, Thomas S; Houser, Carolyn R

    2017-11-01

    While numerous changes in the GABA system have been identified in models of Fragile X Syndrome (FXS), alterations in subunits of the GABA A receptors (GABA A Rs) that mediate tonic inhibition are particularly intriguing. Considering the key role of tonic inhibition in controlling neuronal excitability, reduced tonic inhibition could contribute to FXS-associated disorders such as hyperactivity, hypersensitivity, and increased seizure susceptibility. The current study has focused on the expression and function of the δ subunit of the GABA A R, a major subunit involved in tonic inhibition, in granule cells of the dentate gyrus in the Fmr1 knockout (KO) mouse model of FXS. Electrophysiological studies of dentate granule cells revealed a marked, nearly four-fold, decrease in tonic inhibition in the Fmr1 KO mice, as well as reduced effects of two δ subunit-preferring pharmacological agents, THIP and DS2, supporting the suggestion that δ subunit-containing GABA A Rs are compromised in the Fmr1 KO mice. Immunohistochemistry demonstrated a small but statistically significant decrease in δ subunit labeling in the molecular layer of the dentate gyrus in Fmr1 KO mice compared to wildtype (WT) littermates. The discrepancy between the large deficits in GABA-mediated tonic inhibition in granule cells in the Fmr1 KO mice and only modest reductions in immunolabeling of the δ subunit led to studies of surface expression of the δ subunit. Cross-linking experiments followed by Western blot analysis demonstrated a small, non-significant decrease in total δ subunit protein in the hippocampus of Fmr1 KO mice, but a four-fold decrease in surface expression of the δ subunit in these mice. No significant changes were observed in total or surface expression of the α4 subunit protein, a major partner of the δ subunit in the forebrain. Postembedding immunogold labeling for the δ subunit demonstrated a large, three-fold, decrease in the number of symmetric synapses with

  4. Ethanol activation of protein kinase A regulates GABA-A receptor subunit expression in the cerebral cortex and contributes to ethanol-induced hypnosis

    Directory of Open Access Journals (Sweden)

    Sandeep eKumar

    2012-04-01

    Full Text Available Protein kinases are implicated in neuronal cell functions such as modulation of ion channel function, trafficking and synaptic excitability. Both protein kinase C (PKC and A (PKA are involved in regulation of γ-aminobutyric acid type A (GABA-A receptors through phosphorylation. However, the role of PKA in regulating GABA-A receptors following acute ethanol exposure is not known. The present study investigated the role of PKA in ethanol effects on GABA-A receptor α1 subunit expression in the P2 synaptosomal fraction of the rat cerebral cortex. Additionally, GABA-related behaviors were also examined. Rats were administered ethanol (2.0 – 3.5 g/kg or saline and PKC, PKA and GABA-A receptor α1 subunit levels were measured by Western blot analysis. Ethanol (3.5 g/kg transiently increased GABA-A receptor α1 subunit expression and PKA RIIβ subunit expression at similar time points whereas PKA RIIα was increased at later time points. In contrast, PKC isoform expression remained unchanged. Notably, the moderate ethanol dose (2.0g/kg had no effect on GABA-A α1 subunit levels although PKA RIIα and RIIβ were increased at 10 and 60 minutes, when PKC isozymes are also known to be elevated. To determine if PKA activation was responsible for the ethanol-induced elevation of GABA-A α1 subunits, the PKA antagonist H89 was administered to rats prior to ethanol exposure. H89 administration prevented ethanol-induced increases in GABA-A receptor α1 subunit expression. Moreover, increasing PKA activity intracerebroventricularly with Sp-cAMP prior to a hypnotic dose of ethanol increased ethanol-induced loss of righting reflex duration. This effect appears to be mediated in part by GABA-A receptors as increasing PKA activity also increased the duration of muscimol-induced loss of righting reflex. Overall these data suggest that PKA mediates ethanol-induced GABA-A receptor expression and contributes to ethanol behavioral effects involving GABA-A receptors.

  5. Acetylcholine Receptor: Complex of Homologous Subunits

    Science.gov (United States)

    Raftery, Michael A.; Hunkapiller, Michael W.; Strader, Catherine D.; Hood, Leroy E.

    1980-06-01

    The acetylcholine receptor from the electric ray Torpedo californica is composed of five subunits; two are identical and the other three are structurally related to them. Microsequence analysis of the four polypeptides demonstrates amino acid homology among the subunits. Further sequence analysis of both membrane-bound and Triton-solubilized, chromatographically purified receptor gave the stoichiometry of the four subunits (40,000:50,000:60,000:65,000 daltons) as 2:1:1:1, indicating that this protein is a pentameric complex with a molecular weight of 255,000 daltons. Genealogical analysis suggests that divergence from a common ancestral gene occurred early in the evolution of the receptor. This shared ancestry argues that each of the four subunits plays a functional role in the receptor's physiological action.

  6. Distinct Subunit Domains Govern Synaptic Stability and Specificity of the Kainate Receptor

    Directory of Open Access Journals (Sweden)

    Christoph Straub

    2016-07-01

    Full Text Available Synaptic communication between neurons requires the precise localization of neurotransmitter receptors to the correct synapse type. Kainate-type glutamate receptors restrict synaptic localization that is determined by the afferent presynaptic connection. The mechanisms that govern this input-specific synaptic localization remain unclear. Here, we examine how subunit composition and specific subunit domains contribute to synaptic localization of kainate receptors. The cytoplasmic domain of the GluK2 low-affinity subunit stabilizes kainate receptors at synapses. In contrast, the extracellular domain of the GluK4/5 high-affinity subunit synergistically controls the synaptic specificity of kainate receptors through interaction with C1q-like proteins. Thus, the input-specific synaptic localization of the native kainate receptor complex involves two mechanisms that underlie specificity and stabilization of the receptor at synapses.

  7. Basal Levels of AMPA Receptor GluA1 Subunit Phosphorylation at Threonine 840 and Serine 845 in Hippocampal Neurons

    Science.gov (United States)

    Babiec, Walter E.; Guglietta, Ryan; O'Dell, Thomas J.

    2016-01-01

    Dephosphorylation of AMPA receptor (AMPAR) GluA1 subunits at two sites, serine 845 (S845) and threonine 840 (T840), is thought to be involved in NMDA receptor-dependent forms of long-term depression (LTD). Importantly, the notion that dephosphorylation of these sites contributes to LTD assumes that a significant fraction of GluA1 subunits are…

  8. Fc receptor gamma subunit polymorphisms and systemic lupus erythematosus

    International Nuclear Information System (INIS)

    Al-Ansari, Aliya; Ollier, W.E.; Gonzalez-Gay, Miguel A.; Gul, Ahmet; Inanac, Murat; Ordi, Jose; Teh, Lee-Suan; Hajeer, Ali H.

    2004-01-01

    To investigate the possible association between Fc receptor gamma polymorphisms and systemic lupus erythematosus (SLE). We have investigated the full FcR gamma gene for polymorphisms using polymerase chain reaction (PCR)-single strand confirmational polymorphisms and DNA sequencing .The polymorphisms identified were genotype using PCR-restriction fragment length polymorphism. Systemic lupus erythematosus cases and controls were available from 3 ethnic groups: Turkish, Spanish and Caucasian. The study was conducted in the year 2001 at the Arthritis Research Campaign, Epidemiology Unit, Manchester University Medical School, Manchester, United Kingdom. Five single nucleotide polymorphisms were identified, 2 in the promoter, one in intron 4 and, 2 in the 3'UTR. Four of the 5 single nucleotide polymorphisms (SNPs) were relatively common and investigated in the 3 populations. Allele and genotype frequencies of all 4 investigated SNPs were not statistically different cases and controls. fc receptor gamma gene does not appear to contribute to SLE susceptibility. The identified polymorphisms may be useful in investigating other diseases where receptors containing the FcR gamma subunit contribute to the pathology. (author)

  9. α-4 subunit of nicotinic acetylcholine receptor polymorphisms exhibit ...

    African Journals Online (AJOL)

    Background: Smoking behavior is influenced by both genetic and environmental factors. Nicotine is the major addictive substance in cigarettes. Nicotinic acetylcholine receptors (nAChRs) are thought to play an important role in nicotine addiction of smokers. One of the genes, α-4 subunit of nicotinic acetylcholine receptor ...

  10. Three C-terminal residues from the sulphonylurea receptor contribute to the functional coupling between the KATP channel subunits SUR2A and Kir6.2

    Science.gov (United States)

    Dupuis, Julien P; Revilloud, Jean; Moreau, Christophe J; Vivaudou, Michel

    2008-01-01

    Cardiac ATP-sensitive potassium (KATP) channels are metabolic sensors formed by the association of the inward rectifier potassium channel Kir6.2 and the sulphonylurea receptor SUR2A. SUR2A adjusts channel gating as a function of intracellular ATP and ADP and is the target of pharmaceutical openers and blockers which, respectively, up- and down-regulate Kir6.2. In an effort to understand how effector binding to SUR2A translates into Kir6.2 gating modulation, we examined the role of a 65-residue SUR2A fragment linking transmembrane domain TMD2 and nucleotide-binding domain NBD2 that has been shown to interact with Kir6.2. This fragment of SUR2A was replaced by the equivalent residues of its close homologue, the multidrug resistance protein MRP1. The chimeric construct was expressed in Xenopus oocytes and characterized using the patch-clamp technique. We found that activation by MgADP and synthetic openers was greatly attenuated although apparent affinities were unchanged. Further chimeragenetic and mutagenetic studies showed that mutation of three residues, E1305, I1310 and L1313 (rat numbering), was sufficient to confer this defective phenotype. The same mutations had no effects on channel block by the sulphonylurea glibenclamide or by ATP, suggesting a role for these residues in activatory – but not inhibitory – transduction processes. These results indicate that, within the KATP channel complex, the proximal C-terminal of SUR2A is a critical link between ligand binding to SUR2A and Kir6.2 up-regulation. PMID:18450778

  11. Partial agonists and subunit selectivity at NMDA receptors

    DEFF Research Database (Denmark)

    Risgaard, Rune; Hansen, Kasper Bø; Clausen, Rasmus Prætorius

    2010-01-01

    Subunit-selective ligands for glutamate receptors remains an area of interest as glutamate is the major excitatory neurotransmitter in the brain and involved in a number of diseased states in the central nervous system (CNS). Few subtype-selective ligands are known, especially among the N...

  12. Sex Hormone Receptor Expression in the Human Vocal Fold Subunits.

    Science.gov (United States)

    Kirgezen, Tolga; Sunter, Ahmet Volkan; Yigit, Ozgur; Huq, Gulben Erdem

    2017-07-01

    The study aimed to evaluate the existence of sex hormone receptors in the subunits of vocal fold. This is a cadaver study. The androgen, estrogen, and progesterone receptors were examined in the epithelium (EP), superficial layer of the lamina propria (SLP), vocal ligament (VL), and macula flava (MF) of the vocal folds from 42 human cadavers (21 male, 21 female) by immunohistochemical methods. Their staining ratios were scored and statistically compared. The androgen receptor score was significantly higher for the MF than for the EP and SLP (P vocal fold, mostly in the MF and VLs. Copyright © 2017 The Voice Foundation. Published by Elsevier Inc. All rights reserved.

  13. Glycine Receptor α2 Subunit Activation Promotes Cortical Interneuron Migration

    Directory of Open Access Journals (Sweden)

    Ariel Avila

    2013-08-01

    Full Text Available Glycine receptors (GlyRs are detected in the developing CNS before synaptogenesis, but their function remains elusive. This study demonstrates that functional GlyRs are expressed by embryonic cortical interneurons in vivo. Furthermore, genetic disruption of these receptors leads to interneuron migration defects. We discovered that extrasynaptic activation of GlyRs containing the α2 subunit in cortical interneurons by endogenous glycine activates voltage-gated calcium channels and promotes calcium influx, which further modulates actomyosin contractility to fine-tune nuclear translocation during migration. Taken together, our data highlight the molecular events triggered by GlyR α2 activation that control cortical tangential migration during embryogenesis.

  14. Behavioural endophenotypes in mice lacking the auxiliary GABAB receptor subunit KCTD16.

    Science.gov (United States)

    Cathomas, Flurin; Sigrist, Hannes; Schmid, Luca; Seifritz, Erich; Gassmann, Martin; Bettler, Bernhard; Pryce, Christopher R

    2017-01-15

    Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain and is implicated in the pathophysiology of a number of neuropsychiatric disorders. The GABA B receptors are G-protein coupled receptors consisting of principle subunits and auxiliary potassium channel tetramerization domain (KCTD) subunits. The KCTD subunits 8, 12, 12b and 16 are cytosolic proteins that determine the kinetics of the GABA B receptor response. Previously, we demonstrated that Kctd12 null mutant mice (Kctd12 -/- ) exhibit increased auditory fear learning and that Kctd12 +/- mice show altered circadian activity, as well as increased intrinsic excitability in hippocampal pyramidal neurons. KCTD16 has been demonstrated to influence neuronal excitability by regulating GABA B receptor-mediated gating of postsynaptic ion channels. In the present study we investigated for behavioural endophenotypes in Kctd16 -/- and Kctd16 +/- mice. Compared with wild-type (WT) littermates, auditory and contextual fear conditioning were normal in both Kctd16 -/- and Kctd16 +/- mice. When fear memory was tested on the following day, Kctd16 -/- mice exhibited less extinction of auditory fear memory relative to WT and Kctd16 +/- mice, as well as more contextual fear memory relative to WT and, in particular, Kctd16 +/- mice. Relative to WT, both Kctd16 +/- and Kctd16 -/- mice exhibited normal circadian activity. This study adds to the evidence that auxillary KCTD subunits of GABA B receptors contribute to the regulation of behaviours that could constitute endophenotypes for hyper-reactivity to aversive stimuli in neuropsychiatric disorders. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Activity-dependent control of NMDA receptor subunit composition at hippocampal mossy fibre synapses.

    Science.gov (United States)

    Carta, Mario; Srikumar, Bettadapura N; Gorlewicz, Adam; Rebola, Nelson; Mulle, Christophe

    2018-02-15

    CA3 pyramidal cells display input-specific differences in the subunit composition of synaptic NMDA receptors (NMDARs). Although at low density, GluN2B contributes significantly to NMDAR-mediated EPSCs at mossy fibre synapses. Long-term potentiation (LTP) of NMDARs triggers a modification in the subunit composition of synaptic NMDARs by insertion of GluN2B. GluN2B subunits are essential for the expression of LTP of NMDARs at mossy fibre synapses. Single neurons express NMDA receptors (NMDARs) with distinct subunit composition and biophysical properties that can be segregated in an input-specific manner. The dynamic control of the heterogeneous distribution of synaptic NMDARs is crucial to control input-dependent synaptic integration and plasticity. In hippocampal CA3 pyramidal cells from mice of both sexes, we found that mossy fibre (MF) synapses display a markedly lower proportion of GluN2B-containing NMDARs than associative/commissural synapses. The mechanism involved in such heterogeneous distribution of GluN2B subunits is not known. Here we show that long-term potentiation (LTP) of NMDARs, which is selectively expressed at MF-CA3 pyramidal cell synapses, triggers a modification in the subunit composition of synaptic NMDARs by insertion of GluN2B. This activity-dependent recruitment of GluN2B at mature MF-CA3 pyramidal cell synapses contrasts with the removal of GluN2B subunits at other glutamatergic synapses during development and in response to activity. Furthermore, although expressed at low levels, GluN2B is necessary for the expression of LTP of NMDARs at MF-CA3 pyramidal cell synapses. Altogether, we reveal a previously unknown activity-dependent regulation and function of GluN2B subunits that may contribute to the heterogeneous plasticity induction rules in CA3 pyramidal cells. © 2017 Centre Nationnal de la Recherche Scientifique. The Journal of Physiology © 2017 The Physiological Society.

  16. Modulation of NMDA Receptor Properties and Synaptic Transmission by the NR3A Subunit in Mouse Hippocampal and Cerebrocortical Neurons

    Science.gov (United States)

    Tong, Gary; Takahashi, Hiroto; Tu, Shichun; Shin, Yeonsook; Talantova, Maria; Zago, Wagner; Xia, Peng; Nie, Zhiguo; Goetz, Thomas; Zhang, Dongxian; Lipton, Stuart A.; Nakanishi, Nobuki

    2015-01-01

    Expression of the NR3A subunit with NR1/NR2 in Xenopus oocytes or mammalian cell lines leads to a reduction in N-methyl-D-aspartate (NMDA)-induced currents and decreased Mg2+ sensitivity and Ca2+ permeability compared with NR1/NR2 receptors. Consistent with these findings, neurons from NR3A knockout (KO) mice exhibit enhanced NMDA-induced currents. Recombinant NR3A can also form excitatory glycine receptors with NR1 in the absence of NR2. However, the effects of NR3A on channel properties in neurons and synaptic transmission have not been fully elucidated. To study physiological roles of NR3A subunits, we generated NR3A transgenic (Tg) mice. Cultured NR3A Tg neurons exhibited two populations of NMDA receptor (NMDAR) channels, reduced Mg2+ sensitivity, and decreased Ca2+ permeability in response to NMDA/glycine, but glycine alone did not elicit excitatory currents. In addition, NMDAR-mediated excitatory postsynaptic currents (EPSCs) in NR3A Tg hippocampal slices showed reduced Mg2+ sensitivity, consistent with the notion that NR3A subunits incorporated into synaptic NMDARs. To study the function of endogenous NR3A subunits, we compared NMDAR-mediated EPSCs in NR3A KO and WT control mice. In NR3A KO mice, the ratio of the amplitudes of the NMDAR-mediated component to α-amino-3-hydroxy-5-methyl-4-isox-azolepropionic acid receptor-mediated component of the EPSC was significantly larger than that seen in WT littermates. This result suggests that NR3A subunits contributed to the NMDAR-mediated component of the EPSC in WT mice. Taken together, these results show that NR3A subunits contribute to NMDAR responses from both synaptic and extra-synaptic receptors, likely composed of NR1, NR2, and NR3 subunits. PMID:18003876

  17. Antibodies to the α-subunit of insulin receptor from eggs of immunized hens

    International Nuclear Information System (INIS)

    Song, C.; Yu, J.; Bai, D.H.; Hester, P.Y.; Kim, K.

    1985-01-01

    Simple methods for the generation, purification, and assay of antibodies to the α-subunit of insulin receptor from eggs of immunized hen have been described. Chicken antibodies against the α-subunit inhibit insulin binding to the receptor and stimulate glucose oxidation as well as autophosphorylation of the β-subunit. Thus the properties of chicken antibodies are very similar to those of antibodies found in human autoimmune diseases and different from rabbit antibodies obtained against the same antigen

  18. Expression Profile of the Integrin Receptor Subunits in the Guinea Pig Sclera.

    Science.gov (United States)

    Wang, Kevin K; Metlapally, Ravikanth; Wildsoet, Christine F

    2017-06-01

    The ocular dimensional changes in myopia reflect increased scleral remodeling, and in high myopia, loss of scleral integrity leads to biomechanical weakening and continued scleral creep. As integrins, a type of cell surface receptors, have been linked to scleral remodeling, they represent potential targets for myopia therapies. As a first step, this study aimed to characterize the integrin subunits at the messenger RNA level in the sclera of the guinea pig, a more recently added but increasingly used animal model for myopia research. Primers for α and β integrin subunits were designed using NCBI/UCSC Genome Browser and Primer3 software tools. Total RNA was extracted from normal scleral tissue and isolated cultured scleral fibroblasts, as well as liver and lung, as reference tissues, all from guinea pig. cDNA was produced by reverse transcription, PCR was used to amplify products of predetermined sizes, and products were sequenced using standard methods. Guinea pig scleral tissue expressed all known integrin alpha subunits except αD and αE. The latter integrin subunits were also not expressed by cultured guinea pig scleral fibroblasts; however, their expression was confirmed in guinea pig liver. In addition, isolated cultured fibroblasts did not express integrin subunits αL, αM, and αX. This difference between results for cultured cells and intact sclera presumably reflects the presence in the latter of additional cell types. Both guinea pig scleral tissue and isolated scleral fibroblasts expressed all known integrin beta subunits. All results were verified through sequencing. The possible contributions of integrins to scleral remodeling make them plausible targets for myopia prevention. Data from this study will help guide future ex vivo and in vitro studies directed at understanding the relationship between scleral integrins and ocular growth regulation in the guinea pig model for myopia.

  19. Dithiothreitol activation of the insulin receptor/kinase does not involve subunit dissociation of the native α2β2 insulin receptor subunit complex

    International Nuclear Information System (INIS)

    Sweet, L.J.; Wilden, P.A.; Pessin, J.E.

    1986-01-01

    The subunit composition of the dithiothreitol- (DTT) activated insulin receptor/kinase was examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and gel filtration chromatography under denaturing or nondenaturing conditions. Pretreatment of 32 P-labeled insulin receptors with 50 mM DTT followed by gel filtration chromatography in 0.1% SDS demonstrated the dissociation of the α 2 β 2 insulin receptor complex (M/sub r/ 400,000) into the monomeric 95,000 β subunit. In contrast, pretreatment of the insulin receptors with 1-50 mM DTT followed by gel filtration chromatography in 0.1% Triton X-100 resulted in no apparent alteration in mobility compared to the untreated insulin receptors. Resolution of this complex by nonreducing SDS-polyacrylamide gel electrophoresis and autoradiography demonstrated the existence of the α 2 β 2 heterotetrameric complex with essentially no αβ heterodimeric or free monomeric β subunit species present. This suggests that the insulin receptor can reoxidize into the M/sub r/ 400,000 complex after the removal of DTT by gel filtration chromatography. To prevent reoxidation, the insulin receptors were pretreated with 50 mM DTT. Under the conditions the insulin receptors migrated as the M/sub r/ 400,000 α 2 β 2 complex. These results demonstrate that treatment of the insulin receptors with high concentrations of DTT, followed by removal of DTT by gel filtration, results in reoxidation of the reduced α 2 β 2 insulin receptor complex. Further, these results document that although the DTT stimulation of the insulin receptor/kinase does involve reduction of the insulin receptor subunits, it does not result in dissociation of the native α 2 β 2 insulin receptor subunit complex

  20. Editing modifies the GABA(A) receptor subunit alpha3

    DEFF Research Database (Denmark)

    Ohlson, Johan; Pedersen, Jakob Skou; Haussler, David

    2007-01-01

    Adenosine to inosine (A-to-I) pre-mRNA editing by the ADAR enzyme family has the potential to increase the variety of the proteome. This editing by adenosine deamination is essential in mammals for a functional brain. To detect novel substrates for A-to-I editing we have used an experimental method...... to find selectively edited sites and combined it with bioinformatic techniques that find stem-loop structures suitable for editing. We present here the first verified editing candidate detected by this screening procedure. We show that Gabra-3, which codes for the alpha3 subunit of the GABA(A) receptor......, is a substrate for editing by both ADAR1 and ADAR2. Editing of the Gabra-3 mRNA recodes an isoleucine to a methionine. The extent of editing is low at birth but increases with age, reaching close to 100% in the adult brain. We therefore propose that editing of the Gabra-3 mRNA is important for normal brain...

  1. Regulated appearance of NMDA receptor subunits and channel functions during in vitro neuronal differentiation

    NARCIS (Netherlands)

    Jelitai, Márta; Schlett, Katalin; Varju, Patrícia; Eisel, Ulrich; Madarász, Emília

    The schedule of NMDA receptor subunit expression and the appearance of functional NMDA-gated ion channels were investigated during the retinoic acid (RA) induced neuronal differentiation of NE-4C, a p53-deficient mouse neuroectodermal progenitor cell line. NR2A. NR2B, and NR2D subunit transcripts

  2. Nicotinic acetylcholine receptor: subunit structure, functional binding sites, and ion transport properties

    International Nuclear Information System (INIS)

    Raftery, M.A.; Dunn, S.M.J.; Conti-Tronconi, B.M.; Middlemas, D.S.; Crawford, R.D.

    1983-01-01

    The structure of the nicotinic acetylcholine receptor has been highly conserved during animal evolution, and in all the species and tissues studied so far, including mammals, it is a pseudosymmetric, pentameric complex of related subunits with very similar physical properties. All subunits of these nicotinic receptors were derived from a common ancestral gene, probably by way of gene duplications occurring very early in animal evolution. 45 refs., 8 figs., 2 tabs

  3. Structural basis of subunit selectivity for competitive NMDA receptor antagonists with preference for GluN2A over GluN2B subunits

    Energy Technology Data Exchange (ETDEWEB)

    Lind, Genevieve E.; Mou, Tung-Chung; Tamborini, Lucia; Pomper, Martin G.; De Micheli, Carlo; Conti, Paola; Pinto, Andrea; Hansen, Kasper B. (JHU); (Milan); (Montana)

    2017-07-31

    NMDA-type glutamate receptors are ligand-gated ion channels that contribute to excitatory neurotransmission in the central nervous system (CNS). Most NMDA receptors comprise two glycine-binding GluN1 and two glutamate-binding GluN2 subunits (GluN2A–D). We describe highly potent (S)-5-[(R)-2-amino-2-carboxyethyl]-4,5-dihydro-1H-pyrazole-3-carboxylic acid (ACEPC) competitive GluN2 antagonists, of which ST3 has a binding affinity of 52 nM at GluN1/2A and 782 nM at GluN1/2B receptors. This 15-fold preference of ST3 for GluN1/2A over GluN1/2B is improved compared with NVP-AAM077, a widely used GluN2A-selective antagonist, which we show has 11-fold preference for GluN1/2A over GluN1/2B. Crystal structures of the GluN1/2A agonist binding domain (ABD) heterodimer with bound ACEPC antagonists reveal a binding mode in which the ligands occupy a cavity that extends toward the subunit interface between GluN1 and GluN2A ABDs. Mutational analyses show that the GluN2A preference of ST3 is primarily mediated by four nonconserved residues that are not directly contacting the ligand, but positioned within 12 Å of the glutamate binding site. Two of these residues influence the cavity occupied by ST3 in a manner that results in favorable binding to GluN2A, but occludes binding to GluN2B. Thus, we reveal opportunities for the design of subunit-selective competitive NMDA receptor antagonists by identifying a cavity for ligand binding in which variations exist between GluN2A and GluN2B subunits. This structural insight suggests that subunit selectivity of glutamate-site antagonists can be mediated by mechanisms in addition to direct contributions of contact residues to binding affinity.

  4. Short-term sleep deprivation impairs spatial working memory and modulates expression levels of ionotropic glutamate receptor subunits in hippocampus.

    Science.gov (United States)

    Xie, Meilan; Yan, Jie; He, Chao; Yang, Li; Tan, Gang; Li, Chao; Hu, Zhian; Wang, Jiali

    2015-06-01

    Hippocampus-dependent learning memory is sensitive to sleep deprivation (SD). Although the ionotropic glutamate receptors play a vital role in synaptic plasticity and learning and memory, however, whether the expression of these receptor subunits is modulated by sleep loss remains unclear. In the present study, western blotting was performed by probing with specific antibodies against the ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluA1, GluA2, GluA3, and against the N-methyl-d-aspartate (NMDA) glutamate receptor subunits GluN1, GluN2A, GluN2B. In hippocampus, down regulation of surface GluA1 and GluN2A surface expression were observed in both SD groups. However, surface expression level of GluA2, GluA3, GluN1 and GluN2B was significantly up-regulated in 8h-SD rats when compared to the 4h-SD rats. In parallel with the complex changes in AMPA and NMDA receptor subunit expressions, we found the 8h-SD impaired rat spatial working memory in 30-s-delay T-maze task, whereas no impairment of spatial learning was observed in 4h-SD rats. These results indicate that sleep loss alters the relative expression levels of the AMPA and NMDA receptors, thus affects the synaptic strength and capacity for plasticity and partially contributes to spatial memory impairment. Copyright © 2015. Published by Elsevier B.V.

  5. Regulated appearance of NMDA receptor subunits and channel functions during in vitro neuronal differentiation.

    Science.gov (United States)

    Jelitai, Márta; Schlett, Katalin; Varju, Patrícia; Eisel, Ulrich; Madarász, Emília

    2002-04-01

    The schedule of NMDA receptor subunit expression and the appearance of functional NMDA-gated ion channels were investigated during the retinoic acid (RA) induced neuronal differentiation of NE-4C, a p53-deficient mouse neuroectodermal progenitor cell line. NR2A, NR2B, and NR2D subunit transcripts were present in both nondifferentiated and neuronally differentiated cultures, while NR2C subunits were expressed only transiently, during the early period of neural differentiation. Several splice variants of NR1 were detected in noninduced progenitors and in RA-induced cells, except the N1 exon containing transcripts that appeared after the fourth day of induction, when neuronal processes were already formed. NR1 and NR2A subunit proteins were detected both in nondifferentiated progenitor cells and in neurons, while the mature form of NR2B subunit protein appeared only at the time of neuronal process elongation. Despite the early presence of NR1 and NR2A subunits, NMDA-evoked responses could be detected in NE-4C neurons only after the sixth day of induction, coinciding in time with the expression of the mature NR2B subunit. The formation of functional NMDA receptors also coincided with the appearance of synapsin I and synaptophysin. The lag period between the production of the subunits and the onset of channel function suggests that subunits capable of channel formation cannot form functional NMDA receptors until a certain stage of neuronal commitment. Thus, the in vitro neurogenesis by NE-4C cells provides a suitable tool to investigate some inherent regulatory processes involved in the initial maturation of NMDA receptor complexes. Copyright 2002 Wiley Periodicals, Inc.

  6. Block of nicotinic acetylcholine receptors by philanthotoxins is strongly dependent on their subunit composition

    DEFF Research Database (Denmark)

    Kachel, Hamid S; Patel, Rohit N; Franzyk, Henrik

    2016-01-01

    -fold selectivity of PhTX-12 over PhTX-343 for embryonic muscle-type nicotinic acetylcholine receptors (nAChRs) in TE671 cells. We investigated their inhibition of different neuronal nAChR subunit combinations as well as of embryonic muscle receptors expressed in Xenopus oocytes. Whole-cell currents...

  7. Cloning and expression of the human N-methyl-D-aspartate receptor subunit NR3A

    DEFF Research Database (Denmark)

    Eriksson, Maria; Nilsson, Anna; Froelich-Fabre, Susanne

    2002-01-01

    Native N-methyl-D-aspartate (NMDA) receptors are heteromeric assemblies of four or five subunits. The NMDA receptor subunits, NR1, NR2A, NR2B, NR2C, and NR2D have been cloned in several species, including man. The NR3A subunit, which in rodents is predominantly expressed during early development......, seems to function by reducing the NMDA receptor response. The human homologue to the rat NR3A, however, had not been cloned. In order to study the functions of the human NR3A (hNR3A), we have cloned and sequenced the hNR3A. It was found to share 88% of the DNA sequence with the rat gene, corresponding...

  8. Selective increases of AMPA, NMDA and kainate receptor subunit mRNAs in the hippocampus and orbitofrontal cortex but not in prefrontal cortex of human alcoholics

    Directory of Open Access Journals (Sweden)

    Zhe eJin

    2014-01-01

    Full Text Available Glutamate is the main excitatory transmitter in the human brain. Drugs that affect the glutamatergic signaling will alter neuronal excitability. Ethanol inhibits glutamate receptors. We examined the expression level of glutamate receptor subunit mRNAs in human post-mortem samples from alcoholics and compared the results to brain samples from control subjects. RNA from hippocampal dentate gyrus (HP-DG, orbitofrontal cortex (OFC, and dorso-lateral prefrontal cortex (DL-PFC samples from 21 controls and 19 individuals with chronic alcohol dependence were included in the study. Total RNA was assayed using quantitative RT-PCR. Out of the 16 glutamate receptor subunits, mRNAs encoding two AMPA (2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-ylpropanoic acid receptor subunits GluA2 and GluA3; three kainate receptor subunits GluK2, GluK3 and GluK5 and five NMDA (N-methyl-D-aspartate receptor subunits GluN1, GluN2A, GluN2C, GluN2D and GluN3A were significantly increased in the HP-DG region in alcoholics. In the OFC, mRNA encoding the NMDA receptor subunit GluN3A was increased, whereas in the DL-PFC, no differences in mRNA levels were observed. Our laboratory has previously shown that the expression of genes encoding inhibitory GABA-A receptors is altered in the HP-DG and OFC of alcoholics (Jin et al., 2011. Whether the changes in one neurotransmitter system drives changes in the other or if they change independently is currently not known. The results demonstrate that excessive long-term alcohol consumption is associated with altered expression of genes encoding glutamate receptors in a brain region-specific manner. It is an intriguing possibility that genetic predisposition to alcoholism may contribute to these gene expression changes.

  9. Generation of recombinant antibodies to rat GABAA receptor subunits by affinity selection on synthetic peptides.

    Directory of Open Access Journals (Sweden)

    Sujatha P Koduvayur

    Full Text Available The abundance and physiological importance of GABAA receptors in the central nervous system make this neurotransmitter receptor an attractive target for localizing diagnostic and therapeutic biomolecules. GABAA receptors are expressed within the retina and mediate synaptic signaling at multiple stages of the visual process. To generate monoclonal affinity reagents that can specifically recognize GABAA receptor subunits, we screened two bacteriophage M13 libraries, which displayed human scFvs, by affinity selection with synthetic peptides predicted to correspond to extracellular regions of the rat α1 and β2 GABAA subunits. We isolated three anti-β2 and one anti-α1 subunit specific scFvs. Fluorescence polarization measurements revealed all four scFvs to have low micromolar affinities with their cognate peptide targets. The scFvs were capable of detecting fully folded GABAA receptors heterologously expressed by Xenopus laevis oocytes, while preserving ligand-gated channel activity. Moreover, A10, the anti-α1 subunit-specific scFv, was capable of detecting native GABAA receptors in the mouse retina, as observed by immunofluorescence staining. In order to improve their apparent affinity via avidity, we dimerized the A10 scFv by fusing it to the Fc portion of the IgG. The resulting scFv-Fc construct had a Kd of ∼26 nM, which corresponds to an approximately 135-fold improvement in binding, and a lower detection limit in dot blots, compared to the monomeric scFv. These results strongly support the use of peptides as targets for generating affinity reagents to membrane proteins and encourage investigation of molecular conjugates that use scFvs as anchoring components to localize reagents of interest at GABAA receptors of retina and other neural tissues, for studies of receptor activation and subunit structure.

  10. Age-related changes in functional postsynaptic nicotinic acetylcholine receptor subunits in neurons of the laterodorsal tegmental nucleus, a nucleus important in drug addiction.

    Science.gov (United States)

    Christensen, Mark H; Kohlmeier, Kristi A

    2016-03-01

    The earlier an individual initiates cigarette smoking, the higher the likelihood of development of dependency to nicotine, the addictive ingredient in cigarettes. One possible mechanism underlying this higher addiction liability is an ontogenetically differential cellular response induced by nicotine in neurons mediating the reinforcing or euphoric effects of this drug, which could arise from age-related differences in the composition of nicotinic acetylcholine receptor (nAChR) subunits. In the current study, we examined whether the subunit composition of nAChRs differed between neurons within the laterodorsal tegmentum (LDT), a nucleus importantly involved in drug addiction associated behaviours, across two periods of ontogeny in which nicotine-mediated excitatory responses were shown to depend on age. To this end, whole-cell patch-clamp recordings in mouse brain slices from identified LDT neurons, in combination with nAChR subunit-specific receptor antagonists, were conducted. Comparison of the contribution of different nAChR subunits to acetylcholine (ACh)-induced inward currents indicated that the contributions of the β2 and/or β4 and α7 nAChR subunits alter across age. Taken together, we conclude that across a limited ontogenetic period, there is plasticity in the subunit composition of nAChRs in LDT neurons. In addition, our data indicate, for the first time, functional presence of α6 nAChR subunits in LDT neurons within the age ranges studied. Changes in subunit composition of nAChRs across ontogeny could contribute to the age-related differential excitability induced by nicotine. Differences in the subunit composition of nAChRs within the LDT would be expected to contribute to ontogenetic-dependent outflow from the LDT to target regions, which include reward-related circuitry. © 2014 Society for the Study of Addiction.

  11. Distribution of AMPA-type glutamate receptor subunits in the chick visual system

    Directory of Open Access Journals (Sweden)

    Pires R.S.

    1997-01-01

    Full Text Available Several glutamate receptor (GluR subunits have been characterized during the past few years. In the present study, subunit-specific antisera were used to determine the distribution of the AMPA-type glutamate receptor subunits GluR1-4 in retinorecipient areas of the chick brain. Six white leghorn chicks (Gallus gallus, 7-15 days old, unknown sex were deeply anesthetized and perfused with 4% buffered paraformaldehyde and brain sections were stained using immunoperoxidase techniques. The AMPA-type glutamate receptor subunits GluR1, GluR2/3 and GluR4 were present in several retinorecipient areas, with varying degrees of colocalization. For example, perikarya in layers 2, 3, and 5 of the optic tectum contained GluR1, whereas GluR2/3 subunits appeared mainly in neurons of layer 13. The GluR4 subunit was only detected in a few cells of the tectal layer 13. GluR1 and GluR2/3 were observed in neurons of the nucleus geniculatus lateralis ventralis, whereas GluR4 was only present in its neuropil. Somata in the accessory optic nucleus appeared to contain GluR2/3 and GluR4, whereas GluR1 was the dominant subunit in the neuropil of this nucleus. These results suggest that different subpopulations of visual neurons might express different combinations of AMPA-type GluR subunits, which in turn might generate different synaptic responses to glutamate derived from retinal ganglion cell axons

  12. GABAA receptor subunit gene expression in human prefrontal cortex: comparison of schizophrenics and controls

    Science.gov (United States)

    Akbarian, S.; Huntsman, M. M.; Kim, J. J.; Tafazzoli, A.; Potkin, S. G.; Bunney, W. E. Jr; Jones, E. G.; Bloom, F. E. (Principal Investigator)

    1995-01-01

    The prefrontal cortex of schizophrenics is hypoactive and displays changes related to inhibitory, GABAergic neurons, and GABAergic synapses. These changes include decreased levels of glutamic acid decarboxylase (GAD), the enzyme for GABA synthesis, upregulation of muscimol binding, and downregulation of benzodiazepine binding to GABAA receptors. Studies in the visual cortex of nonhuman primates have demonstrated that gene expression for GAD and for several GABAA receptor subunit polypeptides is under control of neuronal activity, raising the possibility that similar mechanisms in the hypoactive prefrontal cortex of schizophrenics may explain the abnormalities in GAD and in GABAA receptor regulation. In the present study, which is the first of its type on human cerebral cortex, levels of mRNAs for six GABAA receptor subunits (alpha 1, alpha 2, alpha 5, beta 1, beta 2, gamma 2) and their laminar expression patterns were analyzed in the prefrontal cortex of schizophrenics and matched controls, using in situ hybridization histochemistry and densitometry. Three types of laminar expression pattern were observed: mRNAs for the alpha 1, beta 2, and gamma 2 subunits, which are the predominant receptor subunits expressed in the mature cortex, were expressed at comparatively high levels by cells of all six cortical layers, but most intensely by cells in lower layer III and layer IV. mRNAs for the alpha 2, alpha 5, and beta 1 subunits were expressed at lower levels; alpha 2 and beta 1 were expressed predominantly by cells in layers II, III, and IV; alpha 5 was expressed predominantly in layers IV, V, and VI. There were no significant changes in overall mRNA levels for any of the receptor subunits in the prefrontal cortex of schizophrenics, and the laminar expression pattern of all six receptor subunit mRNAs did not differ between schizophrenics and controls. Because gene expression for GABAA receptor subunits is not consistently altered in the prefrontal cortex of

  13. γ-Aminobutyric Acid Type B (GABAB) Receptor Internalization Is Regulated by the R2 Subunit*

    Science.gov (United States)

    Hannan, Saad; Wilkins, Megan E.; Dehghani-Tafti, Ebrahim; Thomas, Philip; Baddeley, Stuart M.; Smart, Trevor G.

    2011-01-01

    γ-Aminobutyric acid type B (GABAB) receptors are important for slow synaptic inhibition in the CNS. The efficacy of inhibition is directly related to the stability of cell surface receptors. For GABAB receptors, heterodimerization between R1 and R2 subunits is critical for cell surface expression and signaling, but how this determines the rate and extent of receptor internalization is unknown. Here, we insert a high affinity α-bungarotoxin binding site into the N terminus of the R2 subunit and reveal its dominant role in regulating the internalization of GABAB receptors in live cells. To simultaneously study R1a and R2 trafficking, a new α-bungarotoxin binding site-labeling technique was used, allowing α-bungarotoxin conjugated to different fluorophores to selectively label R1a and R2 subunits. This approach demonstrated that R1a and R2 are internalized as dimers. In heterologous expression systems and neurons, the rates and extents of internalization for R1aR2 heteromers and R2 homomers are similar, suggesting a regulatory role for R2 in determining cell surface receptor stability. The fast internalization rate of R1a, which has been engineered to exit the endoplasmic reticulum, was slowed to that of R2 by truncating the R1a C-terminal tail or by removing a dileucine motif in its coiled-coil domain. Slowing the rate of internalization by co-assembly with R2 represents a novel role for GPCR heterodimerization whereby R2 subunits, via their C terminus coiled-coil domain, mask a dileucine motif on R1a subunits to determine the surface stability of the GABAB receptor. PMID:21724853

  14. Positive modulation of delta-subunit containing GABAA receptors in mouse neurons

    DEFF Research Database (Denmark)

    Vardya, Irina; Hoestgaard-Jensen, Kirsten; Nieto-Gonzalez, Jose Luis

    2012-01-01

    δ-subunit containing extrasynaptic GABA(A) receptors are potential targets for modifying neuronal activity in a range of brain disorders. With the aim of gaining more insight in synaptic and extrasynaptic inhibition, we used a new positive modulator, AA29504, of δ-subunit containing GABA(A) recep......δ-subunit containing extrasynaptic GABA(A) receptors are potential targets for modifying neuronal activity in a range of brain disorders. With the aim of gaining more insight in synaptic and extrasynaptic inhibition, we used a new positive modulator, AA29504, of δ-subunit containing GABA......(A) receptors in mouse neurons in vitro and in vivo. Whole-cell patch-clamp recordings were carried out in the dentate gyrus in mouse brain slices. In granule cells, AA29504 (1 μM) caused a 4.2-fold potentiation of a tonic current induced by THIP (1 μM), while interneurons showed a potentiation of 2.6-fold......-free environment using Ca²⁺ imaging in cultured neurons, AA29504 showed GABA(A) receptor agonism in the absence of agonist. Finally, AA29504 exerted dose-dependent stress-reducing and anxiolytic effects in mice in vivo. We propose that AA29504 potentiates δ-containing GABA(A) receptors to enhance tonic inhibition...

  15. Cigarette smoking during pregnancy regulates the expression of specific nicotinic acetylcholine receptor (nAChR) subunits in the human placenta

    Energy Technology Data Exchange (ETDEWEB)

    Machaalani, R., E-mail: rita.machaalani@sydney.edu.au [Department of Medicine, The University of Sydney, NSW 2006 (Australia); Bosch Institute, The University of Sydney, NSW 2006 (Australia); The Children' s Hospital at Westmead, NSW 2145 (Australia); Ghazavi, E. [Bosch Institute, The University of Sydney, NSW 2006 (Australia); School of Medical Sciences (Pharmacology), The University of Sydney, NSW 2006 (Australia); Hinton, T. [School of Medical Sciences (Pharmacology), The University of Sydney, NSW 2006 (Australia); Waters, K.A. [Department of Medicine, The University of Sydney, NSW 2006 (Australia); The Children' s Hospital at Westmead, NSW 2145 (Australia); Hennessy, A. [School of Medicine, University of Western Sydney, NSW 2751 (Australia); Heart Research Institute, 7 Eliza St Newtown, NSW 2042 (Australia)

    2014-05-01

    Smoking during pregnancy is associated with low birth weight, premature delivery, and neonatal morbidity and mortality. Nicotine, a major pathogenic compound of cigarette smoke, binds to the nicotinic acetylcholine receptors (nAChRs). A total of 16 nAChR subunits have been identified in mammals (9 α, 4 β, and 1 δ, γ and ε subunits). The effect of cigarette smoking on the expression of these subunits in the placenta has not yet been determined, thus constituting the aim of this study. Using RT-qPCR and western blotting, this study investigated all 16 mammalian nAChR subunits in the normal healthy human placenta, and compared mRNA and protein expressions in the placentas from smokers (n = 8) to controls (n = 8). Our data show that all 16 subunit mRNAs are expressed in the normal, non-diseased human placenta and that the expression of α2, α3, α4, α9, β2 and β4 subunits is greater than the other subunits. For mRNA, cigarette smoke exposure was associated with increased expression of the α9 subunit, and decreased expression of the δ subunit. At the protein level, expression of both α9 and δ was increased. Thus, cigarette smoking in pregnancy is sufficient to regulate nAChR subunits in the placenta, specifically α9 and δ subunits, and could contribute to the adverse effects of vasoconstriction and decreased re-epithelialisation (α9), and increased calcification and apoptosis (δ), seen in the placentas of smoking women. - Highlights: • All 16 mammalian nAChR subunits are expressed in the human placenta. • Cigarette smoking increases α9 mRNA and protein in the placenta. • Cigarette smoking decreases δ mRNA but increases δ protein in the placenta.

  16. Cigarette smoking during pregnancy regulates the expression of specific nicotinic acetylcholine receptor (nAChR) subunits in the human placenta

    International Nuclear Information System (INIS)

    Machaalani, R.; Ghazavi, E.; Hinton, T.; Waters, K.A.; Hennessy, A.

    2014-01-01

    Smoking during pregnancy is associated with low birth weight, premature delivery, and neonatal morbidity and mortality. Nicotine, a major pathogenic compound of cigarette smoke, binds to the nicotinic acetylcholine receptors (nAChRs). A total of 16 nAChR subunits have been identified in mammals (9 α, 4 β, and 1 δ, γ and ε subunits). The effect of cigarette smoking on the expression of these subunits in the placenta has not yet been determined, thus constituting the aim of this study. Using RT-qPCR and western blotting, this study investigated all 16 mammalian nAChR subunits in the normal healthy human placenta, and compared mRNA and protein expressions in the placentas from smokers (n = 8) to controls (n = 8). Our data show that all 16 subunit mRNAs are expressed in the normal, non-diseased human placenta and that the expression of α2, α3, α4, α9, β2 and β4 subunits is greater than the other subunits. For mRNA, cigarette smoke exposure was associated with increased expression of the α9 subunit, and decreased expression of the δ subunit. At the protein level, expression of both α9 and δ was increased. Thus, cigarette smoking in pregnancy is sufficient to regulate nAChR subunits in the placenta, specifically α9 and δ subunits, and could contribute to the adverse effects of vasoconstriction and decreased re-epithelialisation (α9), and increased calcification and apoptosis (δ), seen in the placentas of smoking women. - Highlights: • All 16 mammalian nAChR subunits are expressed in the human placenta. • Cigarette smoking increases α9 mRNA and protein in the placenta. • Cigarette smoking decreases δ mRNA but increases δ protein in the placenta

  17. Characterisation of the human NMDA receptor subunit NR3A glycine binding site

    DEFF Research Database (Denmark)

    Nilsson, A; Duan, J; Mo-Boquist, L-L

    2007-01-01

    In this study, we characterise the binding site of the human N-methyl-d-aspartate (NMDA) receptor subunit NR3A. Saturation radioligand binding of the NMDA receptor agonists [(3)H]-glycine and [(3)H]-glutamate showed that only glycine binds to human NR3A (hNR3A) with high affinity (K(d)=535nM (277...

  18. Analysis of NR3A receptor subunits in human native NMDA receptors

    DEFF Research Database (Denmark)

    Nilsson, Anna; Eriksson, Maria; Muly, E Chris

    2007-01-01

    NR3A, representing the third class of NMDA receptor subunits, was first studied in rats, demonstrating ubiquitous expression in the developing central nervous system (CNS), but in the adult mainly expressed in spinal cord and some forebrain nuclei. Subsequent studies showed that rodent and non-human...... primate NR3A expression differs. We have studied the distribution of NR3A in the human CNS and show a widespread distribution of NR3A protein in adult human brain. NR3A mRNA and protein were found in all regions of the cerebral cortex, and also in the subcortical forebrain, midbrain and hindbrain. Only...... very low levels of NR3A mRNA and protein could be detected in homogenized adult human spinal cord, and in situ hybridization showed that expression was limited to ventral motoneurons. We found that NR3A is associated with NR1, NR2A and NR2B in adult human CNS, suggesting the existence of native NR1-NR2...

  19. High Affinity IgE-Fc Receptor alpha and gamma Subunit Interactions

    International Nuclear Information System (INIS)

    Rashid, A.; Housden, J. E. M.; Sabban, S.; Helm, B.

    2014-01-01

    Objective: To explore the relationships between the subunits (alpha, beta and gamma) of the high affinity IgE receptor (Fc and RI) and its ability to mediate transmembrane signaling. Study Design: Experimental study. Place and Duration of Study: Department of Molecular Biology and Biotechnology, University of Sheffield, UK, from 2008 to 2009. Methodology: The approach employed was to create a chimera (human alpha-gamma-gamma) using the extracellular (EC) domain of the human high affinity IgE receptor. The alpha subunit (huFc and RIalpha) of IgE receptor was spliced onto the rodent gamma TM and cytoplasmic domain (CD). This was transfected into the Rat Basophilic Leukemia cell line in order to assess the possibility of selectively activating cells transfected with this single pass construct for antigen induced mediator release. Results: The RBLs cell lines transfected with the huFc and RIalpha/gamma/gamma cDNA constructs were assessed for the cell surface expression of the huFc and RIalpha subunit and the response to the antigenic stimulus by looking for degranulation and intracellular Ca2+ mobilisation. The results obtained showed the absence of huFc and RIalpha subunit expression on the surface of transfected cells as seen by flowcytometric studies, beta-hexosaminidase assays and intracellular calcium mobilisation studies. Conclusion: In the present study the grounds for non-expression of huFc and RIalpha/gamma/gamma cDNA remains elusive but may be due to the fact that the human-rodent chimeric receptors are assembled differently than the endogenous rodent receptors as seen in study in which COS 7 cells were transfected with human/rat chimeric complexes. (author)

  20. Differential expression of AMPA-type glutamate receptor subunits during development of the chick optic tectum

    Directory of Open Access Journals (Sweden)

    Batista S.S.

    2002-01-01

    Full Text Available Glutamate receptors have been often associated with developmental processes. We used immunohistochemical techniques to evaluate the expression of the AMPA-type glutamate receptor (GluR subunits in the chick optic tectum (TeO. Chick embryos from the 5th through the 20th embryonic day (E5-E20 and one-day-old (P1 chicks were used. The three types of immunoreactivity evaluated (GluR1, GluR2/3, and GluR4 had different temporal and spatial expression patterns in the several layers of the TeO. The GluR1 subunit first appeared as moderate staining on E7 and then increased on E9. The mature GluR1 pattern included intense staining only in layer 5 of the TeO. The GluR2/3 subunits presented low expression on E5, which became intense on E7. The staining for GluR2/3 changed to very intense on E14 in tectal layer 13. Staining of layer 13 neurons is the most prominent feature of GluR immunoreactivity in the adult TeO. The GluR4 subunit generally presented the lowest expression starting on E7, which was similar to the adult pattern. Some instances of transient expression of GluR subunits were observed in specific cell populations from E9 through E20. These results demonstrate a differential expression of the GluR subunits in the embryonic TeO, adding information about their possible functions in the developmental processes of the visual system.

  1. Deletion of P2X2 and P2X3 receptor subunits does not alter motility of the mouse colon

    Directory of Open Access Journals (Sweden)

    Matthew DeVries

    2010-03-01

    Full Text Available Purinergic P2X receptors contribute to neurotransmission in the gut. P2X receptors are ligand-gated cation channels that mediate synaptic excitation in subsets of enteric neurons. The present study evaluated colonic motility in vitro and in vivo in wild type (WT and P2X2 and P2X3 subunit knockout (KO mice. The muscarinic receptor agonist, bethanechol (0.3-3 micromolar, caused similar contractions of the longitudinal muscle in colon segments from WT, P2X2 and P2X3 subunit KO mice. Nicotine (1-300 micromolar, acting at neuronal nicotinic receptors, caused similar longitudinal muscle relaxations in colonic segments from WT and P2X2 and P2X3 subunit KO mice. Nicotine-induced relaxations were inhibited by nitro-L-arginine (NLA, 100 micromolar and apamin (0.1 micromolar which block inhibitory neuromuscular transmission. ATP (1-1000 micromolar caused contractions only in the presence of NLA and apamin. ATP-induced contractions were similar in colon segments from WT, P2X2 and P2X3 KO mice. The mouse colon generates spontaneous migrating motor complexes (MMCs in vitro. The MMC frequency was higher in P2X2 KO compared to WT tissues; other parameters of the MMC were similar in colon segments from WT, P2X2 and P2X3 KO mice. 5-Hydroxytryptophan-induced fecal output was similar in WT, P2X2 and P2X3 KO mice. These data indicate that nicotinic receptors are located predominately on inhibitory motor neurons supplying the longitudinal muscle in the mouse colon. P2X2 or P2X3 subunit containing receptors are not localized to motorneurons supplying the longitudinal muscle. Synaptic transmission mediated by P2X2 or P2X3 subunit containing receptors is not required for propulsive motility in the mouse colon.

  2. Unsaturated free fatty acids increase benzodiazepine receptor agonist binding depending on the subunit composition of the GABAA receptor complex.

    Science.gov (United States)

    Witt, M R; Westh-Hansen, S E; Rasmussen, P B; Hastrup, S; Nielsen, M

    1996-11-01

    It has been shown previously that unsaturated free fatty acids (FFAs) strongly enhance the binding of agonist benzodiazepine receptor ligands and GABAA receptor ligands in the CNS in vitro. To investigate the selectivity of this effect, recombinant human GABAA/benzodiazepine receptor complexes formed by different subunit compositions (alpha x beta y gamma 2, x = 1, 2, 3, and 5; y = 1, 2, and 3) were expressed using the baculovirus-transfected Sf9 insect cell system. At 10(-4) M, unsaturated FFAs, particularly arachidonic (20:4) and docosahexaenoic (22:6) acids, strongly stimulated (> 200% of control values) the binding of [3H]flunitrazepam ([3H]FNM) to the alpha 3 beta 2 gamma 2 receptor combination in whole cell preparations. No effect or small increases in levels of unsaturated FFAs on [3H]FNM binding to alpha 1 beta x gamma 2 and alpha 2 beta x gamma 2 receptor combinations were observed, and weak effects (130% of control values) were detected using the alpha 5 beta 2 gamma 2 receptor combination. The saturated FFAs, stearic and palmitic acids, were without effect on [3H]FNM binding to any combination of receptor complexes. The hydroxylated unsaturated FFAs, ricinoleic and ricinelaidic acids, were shown to decrease the binding of [3H]FNM only if an alpha 1 beta 2 gamma 2 receptor combination was used. Given the heterogeneity of the GABAA/ benzodiazepine receptor subunit distribution in the CNS, the effects of FFAs on the benzodiazepine receptor can be assumed to vary at both cellular and regional levels.

  3. Habenular expression of rare missense variants of the β4 nicotinic receptor subunit alters nicotine consumption

    Directory of Open Access Journals (Sweden)

    Marta A Ślimak

    2014-01-01

    Full Text Available The CHRNA5-CHRNA3-CHRNB4 gene cluster, encoding the α5, α3 and β4 nicotinic acetylcholine receptor (nAChR subunits, has been linked to nicotine dependence. The habenulo-interpeduncular (Hb-IPN tract is particularly enriched in α3β4 nAChRs. We recently showed that modulation of these receptors in the medial habenula (MHb in mice altered nicotine consumption. Given that β4 is rate-limiting for receptor activity and that single nucleotide polymorphisms (SNPs in CHRNB4 have been linked to altered risk of nicotine dependence in humans, we were interested in determining the contribution of allelic variants of β4 to nicotine receptor activity in the MHb. We screened for missense SNPs with allele frequencies > 0.0005 and introduced the corresponding substitutions in Chrnb4. Fourteen variants were analyzed by co-expression with α3. We found that β4A90I and β4T374I variants, previously shown to associate with reduced risk of smoking, and an additional variant β4D447Y, significantly increased nicotine-evoked current amplitudes, while β4R348C, the mutation most frequently encountered in sporadic amyotrophic lateral sclerosis (sALS, showed reduced nicotine currents. We employed lentiviruses to express β4 or β4 variants in the MHb. Immunoprecipitation studies confirmed that β4 lentiviral-mediated expression leads to specific upregulation of α3β4 but not β2 nAChRs in the Mhb. Mice injected with the β4-containing virus showed pronounced aversion to nicotine as previously observed in transgenic Tabac mice overexpressing Chrnb4 at endogenous sites including the MHb. Habenular expression of the β4 gain-of-function allele T374I also resulted in strong aversion, while transduction with the β4 loss-of function allele R348C failed to induce nicotine aversion. Altogether, these data confirm the critical role of habenular β4 in nicotine consumption, and identify specific SNPs in CHRNB4 that modify nicotine-elicited currents and alter nicotine

  4. Olanzapine Reverses MK-801-Induced Cognitive Deficits and Region-Specific Alterations of NMDA Receptor Subunits

    Science.gov (United States)

    Liu, Xiao; Li, Jitao; Guo, Chunmei; Wang, Hongli; Sun, Yaxin; Wang, Han; Su, Yun-Ai; Li, Keqing; Si, Tianmei

    2018-01-01

    Cognitive dysfunction constitutes an essential component in schizophrenia for its early presence in the pathophysiology of the disease and close relatedness to life quality of patients. To develop effective treatment of cognitive deficits, it is important to understand their neurobiological causes and to identify potential therapeutic targets. In this study, adopting repeated MK-801 treatment as an animal model of schizophrenia, we investigated whether antipsychotic drugs, olanzapine and haloperidol, can reverse MK-801-induced cognitive deficits and how the reversal processes recruited proteins involved in glutamate neurotransmission in rat medial prefrontal cortex (mPFC) and hippocampus. We found that low-dose chronic MK-801 treatment impaired object-in-context recognition memory and reversal learning in the Morris water maze, leaving reference memory relatively unaffected, and that these cognitive deficits can be partially reversed by olanzapine, not haloperidol, treatment. At the molecular level, chronic MK-801 treatment resulted in the reduction of multiple N-methyl-D-aspartate (NMDA) receptor subunits in rat mPFC and olanzapine, not haloperidol, treatment restored the levels of GluN1 and phosphorylated GluN2B in this region. Taken together, MK-801-induced cognitive deficits may be associated with region-specific changes in NMDA receptor subunits and the reversal of specific NMDA receptor subunits may underlie the cognition-enhancing effects of olanzapine. PMID:29375333

  5. Memory Deficits Induced by Inflammation Are Regulated by α5-Subunit-Containing GABAA Receptors

    Directory of Open Access Journals (Sweden)

    Dian-Shi Wang

    2012-09-01

    Full Text Available Systemic inflammation causes learning and memory deficits through mechanisms that remain poorly understood. Here, we studied the pathogenesis of memory loss associated with inflammation and found that we could reverse memory deficits by pharmacologically inhibiting α5-subunit-containing γ-aminobutyric acid type A (α5GABAA receptors and deleting the gene associated with the α5 subunit. Acute inflammation reduces long-term potentiation, a synaptic correlate of memory, in hippocampal slices from wild-type mice, and this reduction was reversed by inhibition of α5GABAA receptor function. A tonic inhibitory current generated by α5GABAA receptors in hippocampal neurons was increased by the key proinflammatory cytokine interleukin-1β through a p38 mitogen-activated protein kinase signaling pathway. Interleukin-1β also increased the surface expression of α5GABAA receptors in the hippocampus. Collectively, these results show that α5GABAA receptor activity increases during inflammation and that this increase is critical for inflammation-induced memory deficits.

  6. Identification of the A2 adenosine receptor binding subunit by photoaffinity crosslinking

    International Nuclear Information System (INIS)

    Barrington, W.W.; Jacobson, K.A.; Hutchison, A.J.; Williams, M.; Stiles, G.L.

    1989-01-01

    A high-affinity iodinated agonist radioligand for the A2 adenosine receptor has been synthesized to facilitate studies of the A2 adenosine receptor binding subunit. The radioligand 125I-labeled PAPA-APEC (125I-labeled 2-[4-(2-[2-[(4- aminophenyl)methylcarbonylamino]ethylaminocarbonyl]- ethyl)phenyl]ethylamino-5'-N-ethylcarboxamidoadenosine) was synthesized and found to bind to the A2 adenosine receptor in bovine striatal membranes with high affinity (Kd = 1.5 nM) and A2 receptor selectivity. Competitive binding studies reveal the appropriate A2 receptor pharmacologic potency order with 5'-N-ethylcarboxamidoadenosine (NECA) greater than (-)-N6-[(R)-1-methyl- 2-phenylethyl]adenosine (R-PIA) greater than (+)-N6-[(S)-1-methyl-2- phenylethyl]adenosine (S-PIA). Adenylate cyclase assays, in human platelet membranes, demonstrate a dose-dependent stimulation of cAMP production. PAPA-APEC (1 microM) produces a 43% increase in cAMP production, which is essentially the same degree of increase produced by 5'-N- ethylcarboxamidoadenosine (the prototypic A2 receptor agonist). These findings combined with the observed guanine nucleotide-mediated decrease in binding suggest that PAPA-APEC is a full A2 agonist. The A2 receptor binding subunit was identified by photoaffinity-crosslinking studies using 125I-labeled PAPA-APEC and the heterobifunctional crosslinking agent N-succinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH). After covalent incorporation, a single specifically radiolabeled protein with an apparent molecular mass of 45 kDa was observed on NaDodSO4/PAGE/autoradiography. Incorporation of 125I-labeled PAPA-APEC into this polypeptide is blocked by agonists and antagonists with the expected potency for A2 receptors and is decreased in the presence of 10(-4) M guanosine 5'-[beta, gamma-imido]triphosphate

  7. Effects of visual deprivation during brain development on expression of AMPA receptor subunits in rat’s hippocampus

    Directory of Open Access Journals (Sweden)

    Sayyed Alireza Talaei

    2015-06-01

    Conclusion: Dark rearing of rats during critical period of brain development changes the relative expression and also arrangement of both AMPA receptor subunits, GluR1 and GluR2 in the hippocampus, age dependently.

  8. GABAA receptor B subunit expression in the superior frontal cortex of human alcoholics

    International Nuclear Information System (INIS)

    Buckley, S.T.; Dodd, P.R.

    2001-01-01

    Full text: Changes in GABA A receptor pharmacology can be ascribed to alterations in expression of specific GABA A receptor subunits. Ethanol is known to be a potent agonist of the GABA A receptor. Chronic abuse of alcohol in humans results in damage of selective brain regions such as the superior frontal cortex (SFC), leading to neuronal cell loss. Studies in our laboratory 1 and elsewhere 2 have shown differences in expression of a number of GABA A receptor subunits in chronic human alcoholism. This suggests that alterations in GABA A receptor composition may be involved in the pathogenesis of alcoholic brain damage. We analysed the expression of the β 1 ,β 2 and β 3 isoforms of the GABA A receptor by a competitive reverse transcription polymerase chain reaction (RT-PCR) technique, which utilised an internal standard (IS) for quantitation. 35 S-dATP was incorporated to enable visualisation of the PCR products. Human brain tissue was obtained at autopsy and stored in 0.32 M sucrose at -80 deg C. Total RNA was extracted from pathologically susceptible and spared regions, SFC and motor cortex respectively,of 22 control and 22 alcoholic patients. 1 μg of total RNA from each sample was co-amplified with 0.5 pg of IS and a ratio determined. A standard consisting of known amounts of β 1 cRNA titrated against 0.5 pg of IS enabled a standard curve to be generated for quantitation of each unknown sample. The samples were subjected to polyacrylamide gel electrophoresis and the dried gel exposed to a phosphorimager screen. Data analysis was performed using the ImageQuant program. Initial results indicate that there is a reduction in expression of all the β transcripts in alcoholics when compared with controls, which supports the hypothesis that the GABA A receptor is altered by alcohol abuse. Supported by NHMRC. Copyright (2001) Australian Neuroscience Society

  9. Distribution of the a2, a3, and a5 nicotinic acetylcholine receptor subunits in the chick brain

    Directory of Open Access Journals (Sweden)

    Torrão A.S.

    1997-01-01

    Full Text Available Nicotinic acetylcholine receptors (nAChRs are ionotropic receptors comprised of a and ß subunits. These receptors are widely distributed in the central nervous system, and previous studies have revealed specific patterns of localization for some nAChR subunits in the vertebrate brain. In the present study we used immunohistochemical methods and monoclonal antibodies to localize the a2, a3, and a5 nAChR subunits in the chick mesencephalon and diencephalon. We observed a differential distribution of these three subunits in the chick brain, and showed that the somata and neuropil of many central structures contain the a5 nAChR subunit. The a2 and a3 subunits, on the other hand, exhibited a more restricted distribution than a5 and other subunits previously studied, namely a7, a8 and ß2. The patterns of distribution of the different nAChR subunits suggest that neurons in many brain structures may contain several subtypes of nAChRs and that in a few regions one particular subtype may determine the cholinergic nicotinic responses

  10. NMDA Receptor Subunits Change after Synaptic Plasticity Induction and Learning and Memory Acquisition

    Directory of Open Access Journals (Sweden)

    María Verónica Baez

    2018-01-01

    Full Text Available NMDA ionotropic glutamate receptors (NMDARs are crucial in activity-dependent synaptic changes and in learning and memory. NMDARs are composed of two GluN1 essential subunits and two regulatory subunits which define their pharmacological and physiological profile. In CNS structures involved in cognitive functions as the hippocampus and prefrontal cortex, GluN2A and GluN2B are major regulatory subunits; their expression is dynamic and tightly regulated, but little is known about specific changes after plasticity induction or memory acquisition. Data strongly suggest that following appropriate stimulation, there is a rapid increase in surface GluN2A-NMDAR at the postsynapses, attributed to lateral receptor mobilization from adjacent locations. Whenever synaptic plasticity is induced or memory is consolidated, more GluN2A-NMDARs are assembled likely using GluN2A from a local translation and GluN1 from local ER. Later on, NMDARs are mobilized from other pools, and there are de novo syntheses at the neuron soma. Changes in GluN1 or NMDAR levels induced by synaptic plasticity and by spatial memory formation seem to occur in different waves of NMDAR transport/expression/degradation, with a net increase at the postsynaptic side and a rise in expression at both the spine and neuronal soma. This review aims to put together that information and the proposed hypotheses.

  11. Expression of five acetylcholine receptor subunit genes in Brugia malayi adult worms

    Directory of Open Access Journals (Sweden)

    Ben-Wen Li

    2015-12-01

    Full Text Available Acetylcholine receptors (AChRs are required for body movement in parasitic nematodes and are targets of “classical” anthelmintic drugs such as levamisole and pyrantel and of newer drugs such as tribendimidine and derquantel. While neurotransmission explains the effects of these drugs on nematode movement, their effects on parasite reproduction are unexplained. The levamisole AChR type (L-AChRs in Caenorhabditis elegans is comprised of five subunits: Cel-UNC-29, Cel-UNC-38, Cel-UNC-63, Cel-LEV-1 and Cel-LEV-8. The genome of the filarial parasite Brugia malayi contains nine AChRs subunits including orthologues of Cel-unc-29, Cel-unc-38, and Cel-unc-63. We performed in situ hybridization with RNA probes to localize the expression of five AChR genes (Bm1_35890-Bma-unc-29, Bm1_20330-Bma-unc-38, Bm1_38195-Bma-unc-63, Bm1_48815-Bma-acr-26 and Bm1_40515-Bma-acr-12 in B. malayi adult worms. Four of these genes had similar expression patterns with signals in body muscle, developing embryos, spermatogonia, uterine wall adjacent to stretched microfilariae, wall of Vas deferens, and lateral cord. Three L-AChR subunit genes (Bma-unc-29, Bma-unc-38 and Bma-unc-63 were expressed in body muscle, which is a known target of levamisole. Bma-acr-12 was co-expressed with these levamisole subunit genes in muscle, and this suggests that its protein product may form receptors with other alpha subunits. Bma-acr-26 was expressed in male muscle but not in female muscle. Strong expression signals of these genes in early embryos and gametes in uterus and testis suggest that AChRs may have a role in nervous system development of embryogenesis and spermatogenesis. This would be consistent with embryotoxic effects of drugs that target these receptors in filarial worms. Our data show that the expression of these receptor genes is tightly regulated with regard to localization in adult worms and developmental stage in embryos and gametes. These results may help to explain the

  12. Somato-synaptic variation of GABA(A) receptors in cultured murine cerebellar granule cells: investigation of the role of the alpha6 subunit.

    Science.gov (United States)

    Mellor, J R; Wisden, W; Randall, A D

    2000-07-10

    Electrophysiological investigation of cultured cerebellar murine granule cells revealed differences between the GABA(A) receptors at inhibitory synapses and those on the cell body. Specifically, mIPSCs decayed more rapidly than cell body receptors deactivated, the mean single channel conductance at the synapse (32 pS) was greater than that at cell body (21 pS) and only cell body receptors were sensitive to Zn(2+) (150 microM), which depressed response amplitude by 82+/-5% and almost doubled the rate of channel deactivation. The GABA(A) receptor alpha6 subunit is selectively expressed in cerebellar granule cells. Although concentrated at synapses, it is also found on extrasynaptic membranes. Using a mouse line (Deltaalpha6lacZ) lacking this subunit, we investigated its role in the somato-synaptic differences in GABA(A) receptor function. All differences between cell body and synaptic GABA(A) receptors observed in wild-type (WT) granule cells persisted in Deltaalpha6lacZ cells, thus demonstrating that they are not specifically due to the cellular distribution of the alpha6 subunit. However, mIPSCs from WT and Deltaalpha6lacZ cells differed in both their kinetics (faster decay in WT cells) and underlying single channel conductance (32 pS WT, 25 pS Deltaalpha6lacZ). This provides good evidence for a functional contribution of the alpha6 subunit to postsynaptic GABA(A) receptors in these cells. Despite this, deactivation kinetics of mIPSCs in WT and Deltaalpha6lacZ granule cells exhibited similar benzodiazepene (BDZ) sensitivity. This suggests that the enhanced BDZ-induced ataxia seen in Deltaalpha6lacZ mice may reflect physiological activity at extrasynaptic receptors which, unlike those at synapses, display differential BDZ-sensitivity in WT and Deltaalpha6lacZ granule cells (Jones, A.M., Korpi, E.R., McKernan, R.M., Nusser, Z., Pelz, R., Makela, R., Mellor, J.R., Pollard, S., Bahn, S., Stephenson, F.A., Randall, A.D., Sieghart, W., Somogyi, P., Smith, A.J.H., Wisden

  13. Novel TPR-containing subunit of TOM complex functions as cytosolic receptor for Entamoeba mitosomal transport.

    Science.gov (United States)

    Makiuchi, Takashi; Mi-ichi, Fumika; Nakada-Tsukui, Kumiko; Nozaki, Tomoyoshi

    2013-01-01

    Under anaerobic environments, the mitochondria have undergone remarkable reduction and transformation into highly reduced structures, referred as mitochondrion-related organelles (MROs), which include mitosomes and hydrogenosomes. In agreement with the concept of reductive evolution, mitosomes of Entamoeba histolytica lack most of the components of the TOM (translocase of the outer mitochondrial membrane) complex, which is required for the targeting and membrane translocation of preproteins into the canonical aerobic mitochondria. Here we showed, in E. histolytica mitosomes, the presence of a 600-kDa TOM complex composed of Tom40, a conserved pore-forming subunit, and Tom60, a novel lineage-specific receptor protein. Tom60, containing multiple tetratricopeptide repeats, is localized to the mitosomal outer membrane and the cytosol, and serves as a receptor of both mitosomal matrix and membrane preproteins. Our data indicate that Entamoeba has invented a novel lineage-specific shuttle receptor of the TOM complex as a consequence of adaptation to an anaerobic environment.

  14. Autoinactivation of the stargazin-AMPA receptor complex: subunit-dependency and independence from physical dissociation.

    Directory of Open Access Journals (Sweden)

    Artur Semenov

    Full Text Available Agonist responses and channel kinetics of native α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA receptors are modulated by transmembrane accessory proteins. Stargazin, the prototypical accessory protein, decreases desensitization and increases agonist potency at AMPA receptors. Furthermore, in the presence of stargazin, the steady-state responses of AMPA receptors show a gradual decline at higher glutamate concentrations. This "autoinactivation" has been assigned to physical dissociation of the stargazin-AMPA receptor complex and suggested to serve as a protective mechanism against overactivation. Here, we analyzed autoinactivation of GluA1-A4 AMPA receptors (all flip isoform expressed in the presence of stargazin. Homomeric GluA1, GluA3, and GluA4 channels showed pronounced autoinactivation indicated by the bell-shaped steady-state dose response curves for glutamate. In contrast, homomeric GluA2i channels did not show significant autoinactivation. The resistance of GluA2 to autoinactivation showed striking dependence on the splice form as GluA2-flop receptors displayed clear autoinactivation. Interestingly, the resistance of GluA2-flip containing receptors to autoinactivation was transferred onto heteromeric receptors in a dominant fashion. To examine the relationship of autoinactivation to physical separation of stargazin from the AMPA receptor, we analyzed a GluA4-stargazin fusion protein. Notably, the covalently linked complex and separately expressed proteins expressed a similar level of autoinactivation. We conclude that autoinactivation is a subunit and splice form dependent property of AMPA receptor-stargazin complexes, which involves structural rearrangements within the complex rather than any physical dissociation.

  15. M1 muscarinic receptor facilitates cognitive function by interplay with AMPA receptor GluA1 subunit.

    Science.gov (United States)

    Zhao, Lan-Xue; Ge, Yan-Hui; Xiong, Cai-Hong; Tang, Ling; Yan, Ying-Hui; Law, Ping-Yee; Qiu, Yu; Chen, Hong-Zhuan

    2018-03-06

    M1 muscarinic acetylcholine receptors (M1 mAChRs) are the most abundant muscarinic receptors in the hippocampus and have been shown to have procognitive effects. AMPA receptors (AMPARs), an important subtype of ionotropic glutamate receptors, are key components in neurocognitive networks. However, the role of AMPARs in procognitive effects of M1 mAChRs and how M1 mAChRs affect the function of AMPARs remain poorly understood. Here, we found that basal expression of GluA1, a subunit of AMPARs, and its phosphorylation at Ser845 were maintained by M1 mAChR activity. Activation of M1 mAChRs promoted membrane insertion of GluA1, especially to postsynaptic densities. Impairment of hippocampus-dependent learning and memory by antagonism of M1 mAChRs paralleled the reduction of GluA1 expression, and improvement of learning and memory by activation of M1 mAChRs was accompanied by the synaptic insertion of GluA1 and its increased phosphorylation at Ser845. Furthermore, abrogation of phosphorylation of Ser845 residue of GluA1 ablated M1 mAChR-mediated improvement of learning and memory. Taken together, these results show a functional correlation of M1 mAChRs and GluA1 and the essential role of GluA1 in M1 mAChR-mediated cognitive improvement.-Zhao, L.-X., Ge, Y.-H., Xiong, C.-H., Tang, L., Yan, Y.-H., Law, P.-Y., Qiu, Y., Chen, H.-Z. M1 muscarinic receptor facilitates cognitive function by interplay with AMPA receptor GluA1 subunit.

  16. Fast and Slow Inhibition in the Visual Thalamus Is Influenced by Allocating GABAA Receptors with Different γ Subunits

    Directory of Open Access Journals (Sweden)

    Zhiwen Ye

    2017-04-01

    Full Text Available Cell-type specific differences in the kinetics of inhibitory postsynaptic conductance changes (IPSCs are believed to impact upon network dynamics throughout the brain. Much attention has focused on how GABAA receptor (GABAAR α and β subunit diversity will influence IPSC kinetics, but less is known about the influence of the γ subunit. We have examined whether GABAAR γ subunit heterogeneity influences IPSC properties in the thalamus. The γ2 subunit gene was deleted from GABAARs selectively in the dorsal lateral geniculate nucleus (dLGN. The removal of the γ2 subunit from the dLGN reduced the overall spontaneous IPSC (sIPSC frequency across all relay cells and produced an absence of IPSCs in a subset of relay neurons. The remaining slower IPSCs were both insensitive to diazepam and zinc indicating the absence of the γ2 subunit. Because these slower IPSCs were potentiated by methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM, we propose these IPSCs involve γ1 subunit-containing GABAAR activation. Therefore, γ subunit heterogeneity appears to influence the kinetics of GABAAR-mediated synaptic transmission in the visual thalamus in a cell-selective manner. We suggest that activation of γ1 subunit-containing GABAARs give rise to slower IPSCs in general, while faster IPSCs tend to be mediated by γ2 subunit-containing GABAARs.

  17. Generation of functional inhibitory synapses incorporating defined combinations of GABA(A or glycine receptor subunits

    Directory of Open Access Journals (Sweden)

    Christine Laura Dixon

    2015-12-01

    Full Text Available Fast inhibitory neurotransmission in the brain is mediated by wide range of GABAA receptor (GABAAR and glycine receptor (GlyR isoforms, each with different physiological and pharmacological properties. Because multiple isoforms are expressed simultaneously in most neurons, it is difficult to define the properties of inhibitory postsynaptic currents mediated by individual isoforms in vivo. Although recombinant expression systems permit the expression of individual isoforms in isolation, they require exogenous agonist application which cannot mimic the dynamic neurotransmitter profile characteristic of native synapses. We describe a neuron-HEK293 cell co-culture technique for generating inhibitory synapses incorporating defined combinations of GABAAR or GlyR subunits. Primary neuronal cultures, prepared from embryonic rat cerebral cortex or spinal cord, are used to provide presynaptic GABAergic and glycinergic terminals, respectively. When the cultures are mature, HEK293 cells expressing the subunits of interest plus neuroligin 2A are plated onto the neurons, which rapidly form synapses onto HEK293 cells. Patch clamp electrophysiology is then used to analyze the physiological and pharmacological properties of the inhibitory postsynaptic currents mediated by the recombinant receptors. The method is suitable for investigating the kinetic properties or the effects of drugs on inhibitory postsynaptic currents mediated by defined GABAAR or GlyR isoforms of interest, the effects of hereditary disease mutations on the formation and function of both types of synapses, and synaptogenesis and synaptic clustering mechanisms. The entire cell preparation procedure takes 2 – 5 weeks.

  18. Age-Related Differences in NMDA Receptor Subunits of Prenatally Methamphetamine-Exposed Male Rats

    Czech Academy of Sciences Publication Activity Database

    Vrajová, M.; Schutová, B.; Klaschka, Jan; Štěpánková, H.; Řípová, D.; Šlamberová, R.

    2014-01-01

    Roč. 39, č. 11 (2014), s. 2040-2046 ISSN 0364-3190 Grant - others:GA ČR(CZ) GAP303/10/0580; Ministerstvo školství(CZ) CSM 7/CRP/2014; Univerzita Karlova(CZ) Prvouk P34; Univerzita Karlova(CZ) 260045/SVV/2014; Prague Psychiatric Center(CZ) MH CZ–DRO: 00023752 Institutional support: RVO:67985807 Keywords : methamphetamine * in-utero * NMDA receptor subunits * hippocampus Subject RIV: FH - Neurology Impact factor: 2.593, year: 2014

  19. Anxiogenic properties of an inverse agonist selective for α3 subunit-containing GABAA receptors

    OpenAIRE

    Atack, John R; Hutson, Peter H; Collinson, Neil; Marshall, George; Bentley, Graham; Moyes, Christopher; Cook, Susan M; Collins, Ian; Wafford, Keith; McKernan, Ruth M; Dawson, Gerard R

    2005-01-01

    α3IA (6-(4-pyridyl)-5-(4-methoxyphenyl)-3-carbomethoxy-1-methyl-1H-pyridin-2-one) is a pyridone with higher binding and functional affinity and greater inverse agonist efficacy for GABAA receptors containing an α3 rather than an α1, α2 or α5 subunit. If doses are selected that minimise the occupancy at these latter subtypes, then the in vivo effects of α3IA are most probably mediated by the α3 subtype.α3IA has good CNS penetration in rats and mice as measured using a [3H]Ro 15-1788 in vivo bi...

  20. Expression and localization of ionotropic glutamate receptor subunits in the goldfish retina--an in situ hybridization and immunocytochemical study

    NARCIS (Netherlands)

    Vandenbranden, C. A.; Kamphuis, W.; Nunes Cardozo, B.; Kamermans, M.

    2000-01-01

    The expression and distribution of AMPA, kainate and NMDA glutamate receptor subunits was studied in the goldfish retina. For the immunocytochemical localization of the AMPA receptor antisera against GluR2, GluR2/3 and GluR4 were used, and for in situ hybridization rat specific probes for GluR1 and

  1. Synthetic α subunit peptide 125-147 of human nicotinic acetylcholine receptor induces antibodies to native receptor

    International Nuclear Information System (INIS)

    McCormick, D.J.; Griesmann, G.E.; Huang, Z.; Lennon, V.A.

    1986-01-01

    A synthetic peptide corresponding to residues 125-147 of the Torpedo acetylcholine receptor (AChR) α subunit proved to be a major antigenic region of the AChR. Rats inoculated with 50 μg of peptide (T α 125-147) developed T cell immunity and antibodies to native AChR and signs of experimental autoimmune myasthenia gravis. They report the synthesis and preliminary testing of a disulfide-looped peptide comprising residues 125-147 of the human AChR α subunit. Peptide H α 125-147 differs from T α 125-147 at residues 139 (Glu for Gln) and 143 (Ser for Thr). In immunoprecipitation assays, antibodies to Torpedo AChR bound 125 I-labelled Hα 125-147 antibody bound Hα 125-147, but monoclonal antibodies to an immunodominant region of native AChR bound neither Hα 125-147 nor T α 125-147. Rats immunized with H α 125-147 produced anti-mammalian muscle AChR antibodies that induced modulation of AChRs from cultured human myotubes. Thus, region 125-147 of the human AChR α subunit is extracellular in muscle, and is both antigenic and immunogenic. It remains to be determined whether or not autoantibodies to this region may in part cause the weakness or myasthenia gravis in man

  2. Synthetic. cap alpha. subunit peptide 125-147 of human nicotinic acetylcholine receptor induces antibodies to native receptor

    Energy Technology Data Exchange (ETDEWEB)

    McCormick, D.J.; Griesmann, G.E.; Huang, Z.; Lennon, V.A.

    1986-03-05

    A synthetic peptide corresponding to residues 125-147 of the Torpedo acetylcholine receptor (AChR) ..cap alpha.. subunit proved to be a major antigenic region of the AChR. Rats inoculated with 50 ..mu..g of peptide (T ..cap alpha.. 125-147) developed T cell immunity and antibodies to native AChR and signs of experimental autoimmune myasthenia gravis. They report the synthesis and preliminary testing of a disulfide-looped peptide comprising residues 125-147 of the human AChR ..cap alpha.. subunit. Peptide H ..cap alpha.. 125-147 differs from T ..cap alpha.. 125-147 at residues 139 (Glu for Gln) and 143 (Ser for Thr). In immunoprecipitation assays, antibodies to Torpedo AChR bound /sup 125/I-labelled H..cap alpha.. 125-147 antibody bound H..cap alpha.. 125-147, but monoclonal antibodies to an immunodominant region of native AChR bound neither H..cap alpha.. 125-147 nor T ..cap alpha.. 125-147. Rats immunized with H ..cap alpha.. 125-147 produced anti-mammalian muscle AChR antibodies that induced modulation of AChRs from cultured human myotubes. Thus, region 125-147 of the human AChR ..cap alpha.. subunit is extracellular in muscle, and is both antigenic and immunogenic. It remains to be determined whether or not autoantibodies to this region may in part cause the weakness or myasthenia gravis in man.

  3. SH2 domains of the p85 alpha subunit of phosphatidylinositol 3-kinase regulate binding to growth factor receptors.

    Science.gov (United States)

    McGlade, C J; Ellis, C; Reedijk, M; Anderson, D; Mbamalu, G; Reith, A D; Panayotou, G; End, P; Bernstein, A; Kazlauskas, A

    1992-01-01

    The binding of cytoplasmic signaling proteins such as phospholipase C-gamma 1 and Ras GTPase-activating protein to autophosphorylated growth factor receptors is directed by their noncatalytic Src homology region 2 (SH2) domains. The p85 alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase, which associates with several receptor protein-tyrosine kinases, also contains two SH2 domains. Both p85 alpha SH2 domains, when expressed individually as fusion proteins in bacteria, bound stably to the activated beta receptor for platelet-derived growth factor (PDGF). Complex formation required PDGF stimulation and was dependent on receptor tyrosine kinase activity. The bacterial p85 alpha SH2 domains recognized activated beta PDGF receptor which had been immobilized on a filter, indicating that SH2 domains contact autophosphorylated receptors directly. Several receptor tyrosine kinases within the PDGF receptor subfamily, including the colony-stimulating factor 1 receptor and the Steel factor receptor (Kit), also associate with PI 3-kinase in vivo. Bacterially expressed SH2 domains derived from the p85 alpha subunit of PI 3-kinase bound in vitro to the activated colony-stimulating factor 1 receptor and to Kit. We infer that the SH2 domains of p85 alpha bind to high-affinity sites on these receptors, whose creation is dependent on receptor autophosphorylation. The SH2 domains of p85 are therefore primarily responsible for the binding of PI 3-kinase to activated growth factor receptors. Images PMID:1372092

  4. Immunocytochemical and stereological analysis of GABA(B) receptor subunit expression in the rat vestibular nucleus following unilateral vestibular deafferentation.

    Science.gov (United States)

    Zhang, Rong; Ashton, John; Horii, Arata; Darlington, Cynthia L; Smith, Paul F

    2005-03-10

    The process of behavioral recovery that occurs following damage to one vestibular labyrinth, vestibular compensation, has been attributed in part to a down-regulation of GABA(B) receptors in the vestibular nucleus complex (VNC) ipsilateral to the lesion, which could potentially reduce commissural inhibition from the contralateral VNC. In this study, we tested the possibility that this occurs through a decrease in the expression of either the GABA(B1) or GABA(B2) subunits of the GABA(B) receptor. We used Western blotting to quantify the expression of these subunits in the VNC at 10 h and 50 h following unilateral vestibular deafferentation (UVD) or sham surgery in rats. We then used immunocytochemistry and stereological counting methods to estimate the number of neurons expressing these subunits in the MVN at 10 h and 2 weeks following UVD or sham surgery. Compared to sham controls, we found no significant changes in either the expression of the two GABA(B) receptor subunits in the VNC or in the number of MVN neurons expressing these GABA(B) receptor subunits post-UVD. These results suggest that GABA(B) receptor expression does not change substantially in the VNC during the process of vestibular compensation.

  5. Multiple thyrotropin β-subunit and thyrotropin receptor-related genes arose during vertebrate evolution.

    Directory of Open Access Journals (Sweden)

    Gersende Maugars

    Full Text Available Thyroid-stimulating hormone (TSH is composed of a specific β subunit and an α subunit that is shared with the two pituitary gonadotropins. The three β subunits derive from a common ancestral gene through two genome duplications (1R and 2R that took place before the radiation of vertebrates. Analysis of genomic data from phylogenetically relevant species allowed us to identify an additional Tshβ subunit-related gene that was generated through 2R. This gene, named Tshβ2, present in cartilaginous fish, little skate and elephant shark, and in early lobe-finned fish, coelacanth and lungfish, was lost in ray-finned fish and tetrapods. The absence of a second type of TSH receptor (Tshr gene in these species suggests that both TSHs act through the same receptor. A novel Tshβ sister gene, named Tshβ3, was generated through the third genomic duplication (3R that occurred early in the teleost lineage. Tshβ3 is present in most teleost groups but was lostin tedraodontiforms. The 3R also generated a second Tshr, named Tshrb. Interestingly, the new Tshrb was translocated from its original chromosomic position after the emergence of eels and was then maintained in its new position. Tshrb was lost in tetraodontiforms and in ostariophysians including zebrafish although the latter species have two TSHs, suggesting that TSHRb may be dispensable. The tissue distribution of duplicated Tshβs and Tshrs was studied in the European eel. The endocrine thyrotropic function in the eel would be essentially mediated by the classical Tshβ and Tshra, which are mainly expressed in the pituitary and thyroid, respectively. Tshβ3 and Tshrb showed a similar distribution pattern in the brain, pituitary, ovary and adipose tissue, suggesting a possible paracrine/autocrine mode of action in these non-thyroidal tissues. Further studies will be needed to determine the binding specificity of the two receptors and how these two TSH systems are interrelated.

  6. Gamma-aminobutyric acid (GABA) stimulates pancreatic cancer growth through overexpressing GABAA receptor pi subunit.

    Science.gov (United States)

    Takehara, Akio; Hosokawa, Masayo; Eguchi, Hidetoshi; Ohigashi, Hiroaki; Ishikawa, Osamu; Nakamura, Yusuke; Nakagawa, Hidewaki

    2007-10-15

    Gamma-aminobutyric acid (GABA) functions primarily as an inhibitory neurotransmitter in the mature central nervous system, and GABA/GABA receptors are also present in nonneural tissues, including cancer, but their precise function in nonneuronal or cancerous cells has thus far been poorly defined. Through the genome-wide cDNA microarray analysis of pancreatic ductal adenocarcinoma (PDAC) cells as well as subsequent reverse transcription-PCR and Northern blot analyses, we identified the overexpression of GABA receptor pi subunit (GABRP) in PDAC cells. We also found the expression of this peripheral type GABAA receptor subunit in few adult human organs. Knockdown of endogenous GABRP expression in PDAC cells by small interfering RNA attenuated PDAC cell growth, suggesting its essential role in PDAC cell viability. Notably, the addition of GABA into the cell culture medium promoted the proliferation of GABRP-expressing PDAC cells, but not GABRP-negative cells, and GABAA receptor antagonists inhibited this growth-promoting effect by GABA. The HEK293 cells constitutively expressing exogenous GABRP revealed the growth-promoting effect of GABA treatment. Furthermore, GABA treatment in GABRP-positive cells increased intracellular Ca2+ levels and activated the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/Erk) cascade. Clinical PDAC tissues contained a higher level of GABA than normal pancreas tissues due to the up-regulation of glutamate decarboxylase 1 expression, suggesting their autocrine/paracrine growth-promoting effect in PDACs. These findings imply that GABA and GABRP could play important roles in PDAC development and progression, and that this pathway can be a promising molecular target for the development of new therapeutic strategies for PDAC.

  7. Functional labeling of insulin receptor subunits in live cells. Alpha 2 beta 2 species is the major autophosphorylated form

    International Nuclear Information System (INIS)

    Le Marchand-Brustel, Y.; Ballotti, R.; Gremeaux, T.; Tanti, J.F.; Brandenburg, D.; Van Obberghen, E.

    1989-01-01

    Both receptor subunits were functionally labeled in order to provide methods allowing, in live cells and in broken cell systems, concomitant evaluation of the insulin receptor dual function, hormone binding, and kinase activity. In cell-free systems, insulin receptors were labeled on their alpha-subunit with 125I-photoreactive insulin, and on their beta-subunit by autophosphorylation. Thereafter, phosphorylated receptors were separated from the complete set of receptors by means of anti-phosphotyrosine antibodies. Using this approach, a subpopulation of receptors was found which had bound insulin, but which were not phosphorylated. Under nonreducing conditions, receptors appeared in three oligomeric species identified as alpha 2 beta 2, alpha 2 beta, and alpha 2. Mainly the alpha 2 beta 2 receptor species was found to be phosphorylated while insulin was bound to alpha 2 beta 2, alpha 2 beta, and alpha 2 forms. In live cells, biosynthetic labeling of insulin receptors was used. Receptors were first labeled with [35S]methionine. Subsequently, the addition of insulin led to receptor autophosphorylation by virtue of the endogenous ATP pool. The total amount of [35S]methionine-labeled receptors was precipitated with antireceptor antibodies, whereas with anti-phosphotyrosine antibodies, only the phosphorylated receptors were isolated. Using this approach we made the two following key findings: (1) Both receptor species, alpha 2 beta 2 and alpha 2 beta, are present in live cells and in comparable amounts. This indicates that the alpha 2 beta form is not a degradation product of the alpha 2 beta 2 form artificially generated during receptor preparation. (2) The alpha 2 beta 2 species is the prevalently autophosphorylated form

  8. Identification and cloning of a gamma 3 subunit splice variant of the human GABA(A) receptor.

    Science.gov (United States)

    Poulsen, C F; Christjansen, K N; Hastrup, S; Hartvig, L

    2000-05-31

    cDNA sequences encoding two forms of the GABA(A) gamma 3 receptor subunit were cloned from human hippocampus. The nucleotide sequences differ by the absence (gamma 3S) or presence (gamma 3L) of 18 bp located in the presumed intracellular loop between transmembrane region (TM) III and IV. The extra 18 bp in the gamma 3L subunit generates a consensus site for phosphorylation by protein kinase C (PKC). Analysis of human genomic DNA encoding the gamma 3 subunit reveals that the 18 bp insert is contiguous with the upstream proximal exon.

  9. Early continuous white noise exposure alters l-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunit glutamate receptor 2 and gamma-aminobutyric acid type a receptor subunit beta3 protein expression in rat auditory cortex.

    Science.gov (United States)

    Xu, Jinghong; Yu, Liping; Zhang, Jiping; Cai, Rui; Sun, Xinde

    2010-02-15

    Auditory experience during the postnatal critical period is essential for the normal maturation of auditory function. Previous studies have shown that rearing infant rat pups under conditions of continuous moderate-level noise delayed the emergence of adult-like topographic representational order and the refinement of response selectivity in the primary auditory cortex (A1) beyond normal developmental benchmarks and indefinitely blocked the closure of a brief, critical-period window. To gain insight into the molecular mechanisms of these physiological changes after noise rearing, we studied expression of the AMPA receptor subunit GluR2 and GABA(A) receptor subunit beta3 in the auditory cortex after noise rearing. Our results show that continuous moderate-level noise rearing during the early stages of development decreases the expression levels of GluR2 and GABA(A)beta3. Furthermore, noise rearing also induced a significant decrease in the level of GABA(A) receptors relative to AMPA receptors. However, in adult rats, noise rearing did not have significant effects on GluR2 and GABA(A)beta3 expression or the ratio between the two units. These changes could have a role in the cellular mechanisms involved in the delayed maturation of auditory receptive field structure and topographic organization of A1 after noise rearing. Copyright 2009 Wiley-Liss, Inc.

  10. High-resolution immunogold localization of AMPA type glutamate receptor subunits at synaptic and non-synaptic sites in rat hippocampus.

    Science.gov (United States)

    Baude, A; Nusser, Z; Molnár, E; McIlhinney, R A; Somogyi, P

    1995-12-01

    particles for the GluRA, GluRB/C and GluRD subunits were present at type 1 synaptic membrane specializations on dendritic spines of pyramidal cells throughout all layers of the CA1 and CA3 areas. The most densely labelled synapses tended to be on the largest spines and many smaller spines remained unlabelled. Immunoparticle density at type 1 synapses on dendritic shafts of some non-principal cells was consistently higher than at labelled synapses of dendritic spines of pyramidal cells. Synapses established between dendritic spines and mossy fibre terminals, were immunoreactive for all studied subunits in stratum lucidum of the CA3 area. The postembedding immunogold method revealed that the AMPA type receptors are concentrated within the main body of the anatomically defined type 1 (asymmetrical) synaptic junction. Often only a part of the membrane specialization showed clustered immunoparticles. There was a sharp decrease in immunoreactive receptor density at the edge of the synaptic specialization. Immunolabelling was consistently demonstrated at extrasynaptic sites on dendrites, dendritic spines and somata. The results demonstrate that the GluRA, B/C and D subunits of the AMPA type glutamate receptor are present in many of the glutamatergic synapses formed by the entorhinal, CA3 pyramidal and mossy fibre terminals. Some interneurons have a higher density of AMPA type receptors in their asymmetrical afferent synapses than pyramidal cells. This may contribute to a lower activation threshold of interneurons as compared to principal cells by the same afferents in the hippocampal formation.

  11. Dopamine receptor D5 deficiency results in a selective reduction of hippocampal NMDA receptor subunit NR2B expression and impaired memory.

    Science.gov (United States)

    Moraga-Amaro, Rodrigo; González, Hugo; Ugalde, Valentina; Donoso-Ramos, Juan Pablo; Quintana-Donoso, Daisy; Lara, Marcelo; Morales, Bernardo; Rojas, Patricio; Pacheco, Rodrigo; Stehberg, Jimmy

    2016-04-01

    Pharmacological evidence associates type I dopamine receptors, including subtypes D1 and D5, with learning and memory. Analyses using genetic approaches have determined the relative contribution of dopamine receptor D1 (D1R) in cognitive tasks. However, the lack of drugs that can discriminate between D1R and D5R has made the pharmacological distinction between the two receptors difficult. Here, we aimed to determine the role of D5R in learning and memory. In this study we tested D5R knockout mice and wild-type littermates in a battery of behavioral tests, including memory, attention, locomotion, anxiety and motivational evaluations. Our results show that genetic deficiency of D5R significantly impairs performance in the Morris water maze paradigm, object location and object recognition memory, indicating a relevant role for D5R in spatial memory and recognition memory. Moreover, the lack of D5R resulted in decreased exploration and locomotion. In contrast, D5R deficiency had no impact on working memory, anxiety and depressive-like behavior, measured using the spontaneous alternation, open-field, tail suspension test, and forced swimming test. Electrophysiological analyses performed on hippocampal slices showed impairment in long-term-potentiation in mice lacking D5R. Further analyses at the molecular level showed that genetic deficiency of D5R results in a strong and selective reduction in the expression of the NMDA receptor subunit NR2B in the hippocampus. These findings demonstrate the relevant contribution of D5R in memory and suggest a functional interaction of D5R with hippocampal glutamatergic pathways. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Linking GABA(A) receptor subunits to alcohol-induced conditioned taste aversion and recovery from acute alcohol intoxication.

    Science.gov (United States)

    Blednov, Y A; Benavidez, J M; Black, M; Chandra, D; Homanics, G E; Rudolph, U; Harris, R A

    2013-04-01

    GABA type A receptors (GABA(A)-R) are important for ethanol actions and it is of interest to link individual subunits with specific ethanol behaviors. We studied null mutant mice for six different GABA(A)-R subunits (α1, α2, α3, α4, α5 and δ). Only mice lacking the α2 subunit showed reduction of conditioned taste aversion (CTA) to ethanol. These results are in agreement with data from knock-in mice with mutation of the ethanol-sensitive site in the α2-subunit (Blednov et al., 2011). All together, they indicate that aversive property of ethanol is dependent on ethanol action on α2-containing GABA(A)-R. Deletion of the α2-subunit led to faster recovery whereas absence of the α3-subunit slowed recovery from ethanol-induced incoordination (rotarod). Deletion of the other four subunits did not affect this behavior. Similar changes in this behavior for the α2 and α3 null mutants were found for flurazepam motor incoordination. However, no differences in recovery were found in motor-incoordinating effects of an α1-selective modulator (zolpidem) or an α4-selective agonist (gaboxadol). Therefore, recovery of rotarod incoordination is under control of two GABA(A)-R subunits: α2 and α3. For motor activity, α3 null mice demonstrated higher activation by ethanol (1 g/kg) whereas both α2 (-/-) and α3 (-/Y) knockout mice were less sensitive to ethanol-induced reduction of motor activity (1.5 g/kg). These studies demonstrate that the effects of ethanol at GABAergic synapses containing α2 subunit are important for specific behavioral effects of ethanol which may be relevant to the genetic linkage of the α2 subunit with human alcoholism. Copyright © 2012 Elsevier Ltd. All rights reserved.

  13. LINKING GABAA RECEPTOR SUBUNITS TO ALCOHOL-INDUCED CONDITIONED TASTE AVERSION AND RECOVERY FROM ACUTE ALCOHOL INTOXICATION

    Science.gov (United States)

    Blednov, Y.A.; Benavidez, J.M.; Black, M.; Chandra, D.; Homanics, G.E.; Rudolph, U.; Harris, R.A.

    2012-01-01

    GABA type A receptors (GABAA-R) are important for ethanol actions and it is of interest to link individual subunits with specific ethanol behaviors. We studied null mutant mice for six different GABAA-R subunits (α1, α2, α3, α4, α5 and δ). Only mice lacking the α2 subunit showed reduction of conditioned taste aversion (CTA) to ethanol. These results are in agreement with data from knock-in mice with mutation of the ethanol-sensitive site in the α2-subunit (Blednov et al., 2011) and indicate this aversive property of ethanol is dependent on ethanol action on α2-containing GABAA-R. Deletion of the α2-subunit led to faster recovery whereas absence of the α3-subunit slowed recovery from ethanol-induced incoordination (rotarod). Deletion of the other four subunits did not affect this behavior. Similar changes in this behavior for the α2 and α3 null mutants were found for flurazepam motor-incoordination. However, no differences in recovery were found in motor-incoordinating effects of an α1-selective modulator (zolpidem) or an α4-selective agonist (gaboxadol). Therefore, recovery of rotarod incoordination is under control of two GABAA-R subunits: α2 and α3. For motor activity, α3 null mice demonstrated higher activation by ethanol (1 g/kg) whereas both α2 and α3 (-/-) knockout mice were less sensitive to ethanol-induced reduction of motor activity (1.5 g/kg). These studies demonstrate that the effects of ethanol at GABAergic synapses containing α2 subunit are important for specific behavioral effects of ethanol which may be relevant to the genetic linkage of the α2 subunit with human alcoholism. PMID:23147414

  14. Identification of the D-1 dopamine receptor subunit in rat striatum after photoaffinity labeling

    Energy Technology Data Exchange (ETDEWEB)

    Kuno, T; Tanaka, C [Kobe Univ. (Japan). School of Medicine

    1982-12-28

    When rat striatal membranes, photolabeled with (/sup 3/H)dopamine under assay conditions similar to those used for dopamine-sensitive adenylate cyclase, were subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, several radioactively labeled bands appeared. Labeling of these bands was reduced in the presence of non-radioactive dopamine during photolysis, but was unaffected by the presence of sulpiride. Haloperidol preferentially reduced the labeling of the main band which had a molecular weight of about 57,000 rather than the other weakly labeled bands. Labeling of this 57,000 dalton protein was not apparent when rat cerebellar membranes were used and was markedly eliminated by kainic acid-induced lesions that destroyed the intrastriatal nerve cell bodies. These results indicate that this 57,000 dalton protein is the binding subunit of the D-1 dopamine receptor.

  15. Synaptic Changes in AMPA Receptor Subunit Expression in Cortical Parvalbumin Interneurons in the Stargazer Model of Absence Epilepsy

    Directory of Open Access Journals (Sweden)

    Nadia K. Adotevi

    2017-12-01

    Full Text Available Feedforward inhibition is essential to prevent run away excitation within the brain. Recent evidence suggests that a loss of feed-forward inhibition in the corticothalamocortical circuitry may underlie some absence seizures. However, it is unclear if this aberration is specifically linked to loss of synaptic excitation onto local fast-spiking parvalbumin-containing (PV+ inhibitory interneurons, which are responsible for mediating feedforward inhibition within cortical networks. We recently reported a global tissue loss of AMPA receptors (AMPARs, and a specific mistrafficking of these AMPARs in PV+ interneurons in the stargazer somatosensory cortex. The current study was aimed at investigating if cellular changes in AMPAR expression were translated into deficits in receptors at specific synapses in the feedforward inhibitory microcircuit. Using western blot immunolabeling on biochemically isolated synaptic fractions, we demonstrate a loss of AMPAR GluA1–4 subunits in the somatosensory cortex of stargazers compared to non-epileptic control mice. Furthermore, using double post-embedding immunogold-cytochemistry, we show a loss of GluA1–4-AMPARs at excitatory synapses onto cortical PV+ interneurons. Altogether, these data indicate a loss of synaptic AMPAR-mediated excitation of cortical PV+ inhibitory neurons. As the cortex is considered the site of initiation of spike wave discharges (SWDs within the corticothalamocortical circuitry, loss of AMPARs at cortical PV+ interneurons likely impairs feed-forward inhibitory output, and contributes to the generation of SWDs and absence seizures in stargazers.

  16. Differentiated human midbrain-derived neural progenitor cells express excitatory strychnine-sensitive glycine receptors containing α2β subunits.

    Directory of Open Access Journals (Sweden)

    Florian Wegner

    Full Text Available BACKGROUND: Human fetal midbrain-derived neural progenitor cells (NPCs may deliver a tissue source for drug screening and regenerative cell therapy to treat Parkinson's disease. While glutamate and GABA(A receptors play an important role in neurogenesis, the involvement of glycine receptors during human neurogenesis and dopaminergic differentiation as well as their molecular and functional characteristics in NPCs are largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here we investigated NPCs in respect to their glycine receptor function and subunit expression using electrophysiology, calcium imaging, immunocytochemistry, and quantitative real-time PCR. Whole-cell recordings demonstrate the ability of NPCs to express functional strychnine-sensitive glycine receptors after differentiation for 3 weeks in vitro. Pharmacological and molecular analyses indicate a predominance of glycine receptor heteromers containing α2β subunits. Intracellular calcium measurements of differentiated NPCs suggest that glycine evokes depolarisations mediated by strychnine-sensitive glycine receptors and not by D-serine-sensitive excitatory glycine receptors. Culturing NPCs with additional glycine, the glycine-receptor antagonist strychnine, or the Na(+-K(+-Cl(- co-transporter 1 (NKCC1-inhibitor bumetanide did not significantly influence cell proliferation and differentiation in vitro. CONCLUSIONS/SIGNIFICANCE: These data indicate that NPCs derived from human fetal midbrain tissue acquire essential glycine receptor properties during neuronal maturation. However, glycine receptors seem to have a limited functional impact on neurogenesis and dopaminergic differentiation of NPCs in vitro.

  17. Neonicotinoid binding, toxicity and expression of nicotinic acetylcholine receptor subunits in the aphid Acyrthosiphon pisum.

    Directory of Open Access Journals (Sweden)

    Emiliane Taillebois

    Full Text Available Neonicotinoid insecticides act on nicotinic acetylcholine receptor and are particularly effective against sucking pests. They are widely used in crops protection to fight against aphids, which cause severe damage. In the present study we evaluated the susceptibility of the pea aphid Acyrthosiphon pisum to the commonly used neonicotinoid insecticides imidacloprid (IMI, thiamethoxam (TMX and clothianidin (CLT. Binding studies on aphid membrane preparations revealed the existence of high and low-affinity binding sites for [3H]-IMI (Kd of 0.16 ± 0.04 nM and 41.7 ± 5.9 nM and for the nicotinic antagonist [125I]-α-bungarotoxin (Kd of 0.008 ± 0.002 nM and 1.135 ± 0.213 nM. Competitive binding experiments demonstrated that TMX displayed a higher affinity than IMI for [125I]-α-bungarotoxin binding sites while CLT affinity was similar for both [125I]-α-bungarotoxin and [3H]-IMI binding sites. Interestingly, toxicological studies revealed that at 48 h, IMI (LC50 = 0.038 µg/ml and TMX (LC50 = 0.034 µg/ml were more toxic than CLT (LC50 = 0.118 µg/ml. The effect of TMX could be associated to its metabolite CLT as demonstrated by HPLC/MS analysis. In addition, we found that aphid larvae treated either with IMI, TMX or CLT showed a strong variation of nAChR subunit expression. Using semi-quantitative PCR experiments, we detected for all insecticides an increase of Apisumα10 and Apisumβ1 expressions levels, whereas Apisumβ2 expression decreased. Moreover, some other receptor subunits seemed to be differently regulated according to the insecticide used. Finally, we also demonstrated that nAChR subunit expression differed during pea aphid development. Altogether these results highlight species specificity that should be taken into account in pest management strategies.

  18. Neonicotinoid Binding, Toxicity and Expression of Nicotinic Acetylcholine Receptor Subunits in the Aphid Acyrthosiphon pisum

    Science.gov (United States)

    Taillebois, Emiliane; Beloula, Abdelhamid; Quinchard, Sophie; Jaubert-Possamai, Stéphanie; Daguin, Antoine; Servent, Denis; Tagu, Denis

    2014-01-01

    Neonicotinoid insecticides act on nicotinic acetylcholine receptor and are particularly effective against sucking pests. They are widely used in crops protection to fight against aphids, which cause severe damage. In the present study we evaluated the susceptibility of the pea aphid Acyrthosiphon pisum to the commonly used neonicotinoid insecticides imidacloprid (IMI), thiamethoxam (TMX) and clothianidin (CLT). Binding studies on aphid membrane preparations revealed the existence of high and low-affinity binding sites for [3H]-IMI (Kd of 0.16±0.04 nM and 41.7±5.9 nM) and for the nicotinic antagonist [125I]-α-bungarotoxin (Kd of 0.008±0.002 nM and 1.135±0.213 nM). Competitive binding experiments demonstrated that TMX displayed a higher affinity than IMI for [125I]-α-bungarotoxin binding sites while CLT affinity was similar for both [125I]-α-bungarotoxin and [3H]-IMI binding sites. Interestingly, toxicological studies revealed that at 48 h, IMI (LC50 = 0.038 µg/ml) and TMX (LC50 = 0.034 µg/ml) were more toxic than CLT (LC50 = 0.118 µg/ml). The effect of TMX could be associated to its metabolite CLT as demonstrated by HPLC/MS analysis. In addition, we found that aphid larvae treated either with IMI, TMX or CLT showed a strong variation of nAChR subunit expression. Using semi-quantitative PCR experiments, we detected for all insecticides an increase of Apisumα10 and Apisumβ1 expressions levels, whereas Apisumβ2 expression decreased. Moreover, some other receptor subunits seemed to be differently regulated according to the insecticide used. Finally, we also demonstrated that nAChR subunit expression differed during pea aphid development. Altogether these results highlight species specificity that should be taken into account in pest management strategies. PMID:24801634

  19. NMDA receptor subunits in the adult rat hippocampus undergo similar changes after 5 minutes in an open field and after LTP induction.

    Directory of Open Access Journals (Sweden)

    Maria Veronica Baez

    Full Text Available NMDA receptor subunits change during development and their synaptic expression is modified rapidly after synaptic plasticity induction in hippocampal slices. However, there is scarce information on subunits expression after synaptic plasticity induction or memory acquisition, particularly in adults. GluN1, GluN2A and GluN2B NMDA receptor subunits were assessed by western blot in 1 adult rats that had explored an open field (OF for 5 minutes, a time sufficient to induce habituation, 2 mature rat hippocampal neuron cultures depolarized by KCl and 3 hippocampal slices from adult rats where long term potentiation (LTP was induced by theta-burst stimulation (TBS. GluN1 and GluN2A, though not GluN2B, were significantly higher 70 minutes--but not 30 minutes--after a 5 minutes session in an OF. GluN1 and GluN2A total immunofluorescence and puncta in neurites increased in cultures, as evaluated 70 minutes after KCl stimulation. Similar changes were found in hippocampal slices 70 minutes after LTP induction. To start to explore underlying mechanisms, hippocampal slices were treated either with cycloheximide (a translation inhibitor or actinomycin D (a transcription inhibitor during electrophysiological assays. It was corroborated that translation was necessary for LTP induction and expression. The rise in GluN1 depends on transcription and translation, while the increase in GluN2A appears to mainly depend on translation, though a contribution of some remaining transcriptional activity during actinomycin D treatment could not be rouled out. LTP effective induction was required for the subunits to increase. Although in the three models same subunits suffered modifications in the same direction, within an apparently similar temporal course, further investigation is required to reveal if they are related processes and to find out whether they are causally related with synaptic plasticity, learning and memory.

  20. Cholinergic cells in the nucleus basalis of mice express the N-methyl-D-aspartate-receptor subunit NR2C and its replacement by the NR2B subunit enhances frontal and amygdaloid acetylcholine levels

    NARCIS (Netherlands)

    De Souza Silva, M. A.; Dolga, Amalia; Pieri, I.; Marchetti, L.; Eisel, U. L. M.; Huston, J. P.; Dere, E.

    2006-01-01

    It is known that glutamatergic and cholinergic systems interact functionally at the level of the cholinergic basal forebrain. The N-methyl-D-aspartate receptor (NMDA-R) is a multiprotein complex composed of NR1, NR2 and/or NR3 subunits. The subunit composition of NMDA-R of cholinergic cells in the

  1. Deletion of the GluA1 AMPA Receptor Subunit Alters the Expression of Short-Term Memory

    Science.gov (United States)

    Sanderson, David J.; Sprengel, Rolf; Seeburg, Peter H.; Bannerman, David M.

    2011-01-01

    Deletion of the GluA1 AMPA receptor subunit selectively impairs short-term memory for spatial locations. We further investigated this deficit by examining memory for discrete nonspatial visual stimuli in an operant chamber. Unconditioned suppression of magazine responding to visual stimuli was measured in wild-type and GluA1 knockout mice.…

  2. Schedule of NMDA receptor subunit expression and functional channel formation in the course of in vitro-induced neurogenesis

    NARCIS (Netherlands)

    Varju, P; Schlett, K; Eisel, U; Madarasz, E

    NE-7C2 neuroectodermal cells derived from forebrain vesicles of p53-deficient mouse embryos (E9) produce neurons and astrocytes in vitro if induced by all-trans retinoic acid. The reproducible morphological stages of neurogenesis were correlated with the expression of various NMDA receptor subunits.

  3. Expression of NMDA receptor subunits in human blood lymphocytes: A peripheral biomarker in online computer game addiction.

    Science.gov (United States)

    Sadat-Shirazi, Mitra-Sadat; Vousooghi, Nasim; Alizadeh, Bentolhoda; Makki, Seyed Mohammad; Zarei, Seyed Zeinolabedin; Nazari, Shahrzad; Zarrindast, Mohammad Reza

    2018-05-23

    Background and aims Repeated performance of some behaviors such as playing computer games could result in addiction. The NMDA receptor is critically involved in the development of behavioral and drug addictions. It has been claimed that the expression level of neurotransmitter receptors in the brain may be reflected in peripheral blood lymphocytes (PBLs). Methods Here, using a real-time PCR method, we have investigated the mRNA expression of GluN2A, GluN2D, GluN3A, and GluN3B subunits of the NMDA receptor in PBLs of male online computer game addicts (n = 25) in comparison with normal subjects (n = 26). Results Expression levels of GluN2A, GluN2D, and GluN3B subunits were not statistically different between game addicts and the control group. However, the mRNA expression of the GluN3A subunit was downregulated in PBLs of game addicts. Discussion and conclusions Transcriptional levels of GluN2A and GluN2D subunits in online computer game addicts are similar to our previously reported data of opioid addiction and are not different from the control group. However, unlike our earlier finding of drug addiction, the mRNA expression levels of GluN3A and GluN3B subunits in PBLs of game addicts are reduced and unchanged, respectively, compared with control subjects. It seems that the downregulated state of the GluN3A subunit of NMDA receptor in online computer game addicts is a finding that deserves more studies in the future to see whether it can serve as a peripheral biomarker in addiction studies, where the researcher wants to rule out the confusing effects of abused drugs.

  4. The essential role of AMPA receptor GluR2 subunit RNA editing in the normal and diseased brain

    Directory of Open Access Journals (Sweden)

    Amanda Lorraine Wright

    2012-04-01

    Full Text Available AMPA receptors are comprised of different combinations of GluR1-GluR4 (also known as GluA1-GluA4 and GluR-A to GluR-D subunits. The GluR2 subunit is subject to Q/R site RNA editing by the ADAR2 enzyme, which converts a codon for glutamine (Q, present in the GluR2 gene, to a codon for arginine (R found in the mRNA. AMPA receptors are calcium (Ca2+-permeable if they contain the unedited GluR2(Q subunit or if they lack the GluR2 subunit. While most AMPA receptors in the brain contain the edited GluR2(R subunit and are therefore Ca2+-impermeable, recent evidence suggests that Ca2+-permeable GluR2-lacking AMPA receptors are important in synaptic plasticity and learning. However, the presence of Ca2+-permeable AMPA receptors containing unedited GluR2 leads to excitotoxic cell loss. Recent studies have indicated that RNA editing of GluR2 is deregulated in diseases, such as amyotrophic lateral sclerosis (ALS, as well in acute neurodegenerative conditions, such as ischemia. More recently, studies have investigated the regulation of RNA editing and possible causes for its deregulation during disease. In this review, we will explore the role of GluR2 RNA editing in the healthy and diseased brain and outline new insights into the mechanisms that control this process.

  5. Effects of ibuprofen on cognition and NMDA receptor subunit expression across aging.

    Science.gov (United States)

    Márquez Loza, Alejandra; Elias, Valerie; Wong, Carmen P; Ho, Emily; Bermudez, Michelle; Magnusson, Kathy R

    2017-03-06

    Age-related declines in long- and short-term memory show relationships to decreases in N-methyl-d-aspartate (NMDA) receptor expression, which may involve inflammation. This study was designed to determine effects of an anti-inflammatory drug, ibuprofen, on cognitive function and NMDA receptor expression across aging. Male C57BL/6 mice (ages 5, 14, 20, and 26months) were fed ibuprofen (375ppm) in NIH31 diet or diet alone for 6weeks prior to testing. Behavioral testing using the Morris water maze showed that older mice performed significantly worse than younger in spatial long-term memory, reversal, and short-term memory tasks. Ibuprofen enhanced overall performance in the short-term memory task, but this appeared to be more related to improved executive function than memory. Ibuprofen induced significant decreases over all ages in the mRNA densities for GluN2B subunit, all GluN1 splice variants, and GluN1-1 splice forms in the frontal cortex and in protein expression of GluN2A, GluN2B and GluN1 C2' cassettes in the hippocampus. GluN1-3 splice form mRNA and C2' cassette protein were significantly increased across ages in frontal lobes of ibuprofen-treated mice. Ibuprofen did not alter expression of pro-inflammatory cytokines IL-1β and TNFα, but did reduce the area of reactive astrocyte immunostaining in frontal cortex of aged mice. Enhancement in executive function showed a relationship to increased GluN1-3 mRNA and decreased gliosis. These findings suggest that inflammation may play a role in executive function declines in aged animals, but other effects of ibuprofen on NMDA receptors appeared to be unrelated to aging or inflammation. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

  6. Requirement of Nicotinic Acetylcholine Receptor Subunit β2 in the Maintenance of Spiral Ganglion Neurons during Aging

    Science.gov (United States)

    Bao, Jianxin; Lei, Debin; Du, Yafei; Ohlemiller, Kevin K.; Beaudet, Arthur L.; Role, Lorna W.

    2008-01-01

    Age-related hearing loss (presbycusis) is a major health concern for the elderly. Loss of spiral ganglion neurons (SGNs), the primary sensory relay of the auditory system, is associated consistently with presbycusis. The causative molecular events responsible for age-related loss of SGNs are unknown. Recent reports directly link age-related neuronal loss in cerebral cortex with the loss of high-affinity nicotine acetylcholine receptors (nAChRs). In cochlea, cholinergic synapses are made by olivocochlear efferent fibers on the outer hair cells that express α9 nAChR subunits and on the peripheral projections of SGNs that express α2, α4 –7, and β2–3 nAChR subunits. A significantly decreased expression of the β2 nAChR subunit in SGNs was found specifically in mice susceptible to presbycusis. Furthermore, mice lacking the β2 nAChR subunit (β2−/−), but not mice lacking the α5 nAChR subunit (α5−/−), have dramatic hearing loss and significant reduction in the number of SGNs. Our findings clearly established a requirement for β2 nAChR subunit in the maintenance of SGNs during aging. PMID:15788760

  7. Sub-unit Specific Regulation of Type-A GABAergic Receptors during Post-Natal Development of the Auditory Cortex

    Directory of Open Access Journals (Sweden)

    Liisa A. Tremere

    2011-01-01

    Full Text Available The GABA-A receptor has been strongly implicated in the organization and function of cortical sensory circuits in the adult mammal. In the present work, changes in the expression patterns of select GABA-A subunits were examined as a function of development. The RNA expression profiles for three subunit types were studied, α1, β2/3 and δ at four developmental time points, (p0, p15, p30 and p90. The o1, β2/3 subunits were present at birth and following a modest increase early in life; mRNA expression for these subunits were found at stable levels throughout life. The expression pattern for the δ subunit showed the most dramatic changes in the number of positive cells as a function of age. In early life, p0 through p15 expression of mRNA for the δ subunit was quite low but increased in later life, p30 and p90. Together these data suggest that much of the potential for inhibitory connectivity is laid down in the pre and early post-natal periods.

  8. Resistance to cycloxaprid in Laodelphax striatellus is associated with altered expression of nicotinic acetylcholine receptor subunits.

    Science.gov (United States)

    Zhang, Yueliang; Han, Yangchun; Yang, Qiong; Wang, Lihua; He, Peng; Liu, Zewen; Li, Zhong; Guo, Huifang; Fang, Jichao

    2018-04-01

    Cycloxaprid is a new oxabridged cis-configuration neonicotinoid insecticide, the resistance development potential and underlying resistance mechanism of which were investigated in the small brown planthopper, Laodelphax striatellus (Fallén), an important agricultural pest of rice. A cycloxaprid-resistant strain (YN-CPD) only achieved 10-fold higher resistance, in contrast to 106-fold higher resistance to buprofezin and 332-fold higher resistance to chlorpyrifos achieved after exposure to similar selection pressure, and the cycloxaprid selected line showed no cross-resistance to the buprofezin and chlorpyrifos-selected resistance strains. Moreover, we identified 10 nicotinic acetylcholine receptor (nAChR) subunits from the transcriptome of L. striatellus, and six segments had open reading frames (ORFs). While we did not find mutations in the nAChR genes of L. striatellus, subunits Lsα1 and Lsβ1 exhibited, respectively, 9.60-fold and 3.36-fold higher expression in the resistant strain, while Lsα8 exhibited 0.44-fold lower expression. Suppression of Lsα1 through ingestion of dsLsα1 led to an increase in susceptibility to cycloxaprid. The findings indicate that resistance to cycloxaprid develops slowly compared with resistance to other chemicals and without cross-resistance to chlorpyrifos or buprofezin; over-expressed Lsα1 is associated with low cycloxaprid resistance levels, but the importance of over-expressed Lsβ1 and reduced expression of Lsα8 could not be excluded. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  9. The Barley Magnesium Chelatase 150-kD Subunit Is Not an Abscisic Acid Receptor1[OA

    Science.gov (United States)

    Müller, André H.; Hansson, Mats

    2009-01-01

    Magnesium chelatase is the first unique enzyme of the chlorophyll biosynthetic pathway. It is composed of three gene products of which the largest is 150 kD. This protein was recently identified as an abscisic acid receptor in Arabidopsis (Arabidopsis thaliana). We have evaluated whether the barley (Hordeum vulgare) magnesium chelatase large subunit, XanF, could be a receptor for the phytohormone. The study involved analysis of recombinant magnesium chelatase protein as well as several induced chlorophyll-deficient magnesium chelatase mutants with defects identified at the gene and protein levels. Abscisic acid had no effect on magnesium chelatase activity and binding to the barley 150-kD protein could not be shown. Magnesium chelatase mutants showed a wild-type response in respect to postgermination growth and stomatal aperture. Our results question the function of the large magnesium chelatase subunit as an abscisic acid receptor. PMID:19176716

  10. Med1 subunit of the mediator complex in nuclear receptor-regulated energy metabolism, liver regeneration, and hepatocarcinogenesis.

    Science.gov (United States)

    Jia, Yuzhi; Viswakarma, Navin; Reddy, Janardan K

    2014-01-01

    Several nuclear receptors regulate diverse metabolic functions that impact on critical biological processes, such as development, differentiation, cellular regeneration, and neoplastic conversion. In the liver, some members of the nuclear receptor family, such as peroxisome proliferator-activated receptors (PPARs), constitutive androstane receptor (CAR), farnesoid X receptor (FXR), liver X receptor (LXR), pregnane X receptor (PXR), glucocorticoid receptor (GR), and others, regulate energy homeostasis, the formation and excretion of bile acids, and detoxification of xenobiotics. Excess energy burning resulting from increases in fatty acid oxidation systems in liver generates reactive oxygen species, and the resulting oxidative damage influences liver regeneration and liver tumor development. These nuclear receptors are important sensors of exogenous activators as well as receptor-specific endogenous ligands. In this regard, gene knockout mouse models revealed that some lipid-metabolizing enzymes generate PPARα-activating ligands, while others such as ACOX1 (fatty acyl-CoA oxidase1) inactivate these endogenous PPARα activators. In the absence of ACOX1, the unmetabolized ACOX1 substrates cause sustained activation of PPARα, and the resulting increase in energy burning leads to hepatocarcinogenesis. Ligand-activated nuclear receptors recruit the multisubunit Mediator complex for RNA polymerase II-dependent gene transcription. Evidence indicates that the Med1 subunit of the Mediator is essential for PPARα, PPARγ, CAR, and GR signaling in liver. Med1 null hepatocytes fail to respond to PPARα activators in that these cells do not show induction of peroxisome proliferation and increases in fatty acid oxidation enzymes. Med1-deficient hepatocytes show no increase in cell proliferation and do not give rise to liver tumors. Identification of nuclear receptor-specific coactivators and Mediator subunits should further our understanding of the complexities of metabolic

  11. [Beta]-Adrenergic Receptor Activation Rescues Theta Frequency Stimulation-Induced LTP Deficits in Mice Expressing C-Terminally Truncated NMDA Receptor GluN2A Subunits

    Science.gov (United States)

    Moody, Teena D.; Watabe, Ayako M.; Indersmitten, Tim; Komiyama, Noboru H.; Grant, Seth G. N.; O'Dell, Thomas J.

    2011-01-01

    Through protein interactions mediated by their cytoplasmic C termini the GluN2A and GluN2B subunits of NMDA receptors (NMDARs) have a key role in the formation of NMDAR signaling complexes at excitatory synapses. Although these signaling complexes are thought to have a crucial role in NMDAR-dependent forms of synaptic plasticity such as long-term…

  12. Characterisation of 5-HT3C, 5-HT3D and 5-HT3E receptor subunits: evolution, distribution and function.

    Science.gov (United States)

    Holbrook, Joanna D; Gill, Catherine H; Zebda, Noureddine; Spencer, Jon P; Leyland, Rebecca; Rance, Kim H; Trinh, Han; Balmer, Gemma; Kelly, Fiona M; Yusaf, Shahnaz P; Courtenay, Nicola; Luck, Jane; Rhodes, Andrew; Modha, Sundip; Moore, Stephen E; Sanger, Gareth J; Gunthorpe, Martin J

    2009-01-01

    The 5-HT(3) receptor is a member of the 'Cys-loop' family of ligand-gated ion channels that mediate fast excitatory and inhibitory transmission in the nervous system. Current evidence points towards native 5-HT(3) receptors originating from homomeric assemblies of 5-HT(3A) or heteromeric assembly of 5-HT(3A) and 5-HT(3B). Novel genes encoding 5-HT(3C), 5-HT(3D), and 5-HT(3E) have recently been described but the functional importance of these proteins is unknown. In the present study, in silico analysis (confirmed by partial cloning) indicated that 5-HT(3C), 5-HT(3D), and 5-HT(3E) are not human-specific as previously reported: they are conserved in multiple mammalian species but are absent in rodents. Expression profiles of the novel human genes indicated high levels in the gastrointestinal tract but also in the brain, Dorsal Root Ganglion (DRG) and other tissues. Following the demonstration that these subunits are expressed at the cell membrane, the functional properties of the recombinant human subunits were investigated using patch clamp electrophysiology. 5-HT(3C), 5-HT(3D), and 5-HT(3E) were all non-functional when expressed alone. Co-transfection studies to determine potential novel heteromeric receptor interactions with 5-HT(3A) demonstrated that the expression or function of the receptor was modified by 5-HT(3C) and 5-HT(3E), but not 5-HT(3D). The lack of distinct effects on current rectification, kinetics or pharmacology of 5-HT(3A) receptors does not however provide unequivocal evidence to support a direct contribution of 5-HT(3C) or 5-HT(3E) to the lining of the ion channel pore of novel heteromeric receptors. The functional and pharmacological contributions of these novel subunits to human biology and diseases such as irritable bowel syndrome for which 5-HT(3) receptor antagonists have major clinical usage, therefore remains to be fully determined.

  13. Tryptic mapping and membrane topology of the benzodiazepine receptor alpha-subunit

    Energy Technology Data Exchange (ETDEWEB)

    Lentes, K.U.; Venter, J.C.

    1986-05-01

    Rat brain membrane benzodiazepine receptors (BZR) were photoaffinity labelled specifically (in presence or absence of 6 ..mu..M clonazepam) with 10 nM /sup 3/H-flunitrazepam (FNZ). Digestion of the FNZ-labelled, membrane-bound BZR with 200 ..mu..g trypsin/mg membrane protein yielded H/sub 2/O-soluble BZR-fragments of molecular mass (M/sub r/) 34, 31, 28, 24, 21, 18, 16, 12, 10 and 7kDa. Because the 34kDa-peptide is the largest fragment containing a FNZ-binding site they conclude that this represents the extracellular domain of the BZR. In the remaining pellet two labelled peptides with M/sub r/ of 44kDa and 28kDa were found that required the use of detergents for their solubilization; they therefore contain the membrane anchoring domain. Digestion of the 0.5% Na-deoxycholate solubilized, intact BZR (M/sub r/ 51kDa) resulted in the same tryptic pattern as the membrane form of the receptor plus two larger fragments of M/sub r/ 45kDa and 40kDa. Arrangement of all tryptic fragments with reference to the FNZ binding site reveals a membrane topology of the BZR alpha-subunit with 67% (34kDa) for the extracellular domain, 21% (11kDa) for the membrane anchoring domain and 12% (6kDa) for a putative cytoplasmic domain. The overlap between some of the labelled fragments suggest that the BZ binding site must be located near the membrane surface of the extracellular domain.

  14. Novel isoforms of the TFIID subunit TAF4 modulate nuclear receptor-mediated transcriptional activity

    International Nuclear Information System (INIS)

    Brunkhorst, Adrian; Neuman, Toomas; Hall, Anita; Arenas, Ernest; Bartfai, Tamas; Hermanson, Ola; Metsis, Madis

    2004-01-01

    The transcription factor TFIID consists of TATA-binding protein (TBP) and TBP-associated factors (TAFs). TAFs are essential for modulation of transcriptional activity but the regulation of TAFs is complex and many important aspects remain unclear. In this study, we have identified and characterized five novel truncated forms of the TFIID subunit TAF4 (TAF II 135). Analysis of the mouse gene structure revealed that all truncations were the results of alternative splicing and resulted in the loss of domains or parts of domains implicated in TAF4 functional interactions. Results from transcriptional assays showed that several of the TAF4 isoforms exerted dominant negative effects on TAF4 activity in nuclear receptor-mediated transcriptional activation. In addition, alternative TAF4 isoforms could be detected in specific cell types. Our results indicate an additional level of complexity in TAF4-mediated regulation of transcription and suggest context-specific roles for these new TAF4 isoforms in transcriptional regulation in vivo

  15. Recombinant human acetylcholine receptor alpha-subunit induces chronic experimental autoimmune myasthenia gravis.

    Science.gov (United States)

    Lennon, V A; Lambert, E H; Leiby, K R; Okarma, T B; Talib, S

    1991-04-01

    A synthetic gene encoding the 210 N-terminal residues of the alpha-subunit of the nicotinic acetylcholine receptor (AChR) of human skeletal muscle was cloned into an inducible expression plasmid to produce a fusion protein in high yield in Escherichia coli. Like native human AChR, the recombinant human alpha 1-210 protein induced AChR-binding, AChR-modulating, and AChR-blocking autoantibodies in rats when injected once intradermally as an emulsion in CFA, with Bordetella pertussis vaccine as supplementary adjuvant. The minimum dose of recombinant protein required to induce biochemical signs of experimental autoimmune myasthenia gravis (EAMG) with 100% incidence was 2.2 micrograms. With 6.6 to 22 micrograms, serum levels of autoantibodies were persistent, and clinically apparent EAMG lasted more than a month. Clinical, electrophysiological, and biochemical indices of EAMG induced by doses of 66 micrograms or more were more uniformly severe and persistent, with 33% fatality. Rats receiving a control extract of E. coli containing plasmid without the alpha 1-210 codon insert, with adjuvants, did not develop autoantibodies or signs of EAMG. This highly reproducible new model of EAMG induced by a recombinant human autoantigen should be valuable for testing Ag-specific immunotherapeutic strategies that might be applicable to treating acquired myasthenia gravis in humans.

  16. Nicotinic Acetylcholine Receptor α4 Subunit Gene Variation Associated with Attention Deficit Hyperactivity Disorder

    Institute of Scientific and Technical Information of China (English)

    HUANG Xuezhu; XU Yong; LI Qianqian; LIU Pozi; YANG Yuan; ZHANG Fuquan; GUO Tianyou; YANG Chuang; GUO Lanting

    2009-01-01

    Previous pharmacological, human genetics, and animal models have implicated the nicotinic ace-tylcholine receptor a4 subunit (CHRNA4) gene in the pathogenesis of attention deficit/hyperactivity disorder (ADHD). The objective of this study is to examine the genetic association between single nucleotide poly-morphisms in the CHRNA4 gene (rs2273502, rs1044396, rs1044397, and rs3827020 loci) and ADHD. Both case-control and family-based designs are used. Children aged 6 to 16 years were interviewed and as-sessed with the children behavior checklist and the revised conner' parent rating scale to identify probands. No significant differences in the frequency distribution of genotypes or alleles were found between the case and control groups. However, further haplotype analyses showed the CCGG haplotype on dsk for ADHD in 164 case-control samples and the standard transmission disequilibrium test analyses suggest that the allele C of rs2273502 was over-transferred in 98 ADHD parent-offspring tdos. These findings suggest that the CHRNA4 gene may play a role in the pathogenesis of ADHD.

  17. Topology characterization of a benzodiazepine-binding beta-rich domain of the GABAA receptor alpha1 subunit.

    Science.gov (United States)

    Xu, Zhiwen; Fang, Shisong; Shi, Haifeng; Li, Hoiming; Deng, Yiqun; Liao, Yinglei; Wu, Jiun-Ming; Zheng, Hui; Zhu, Huaimin; Chen, Hueih-Min; Tsang, Shui Ying; Xue, Hong

    2005-10-01

    Structural investigation of GABAA receptors has been limited by difficulties imposed by its trans-membrane-complex nature. In the present study, the topology of a membrane-proximal beta-rich (MPB) domain in the C139-L269 segment of the receptor alpha1 subunit was probed by mapping the benzodiazepine (BZ)-binding and epitopic sites, as well as fluorescence resonance energy transfer (FRET) analysis. Ala-scanning and semiconservative substitutions within this segment revealed the contribution of the phenyl rings of Y160 and Y210, the hydroxy group of S186 and the positive charge on R187 to BZ-binding. FRET with the bound BZ ligand indicated the proximity of Y160, S186, R187, and S206 to the BZ-binding site. On the other hand, epitope-mapping using the monoclonal antibodies (mAbs) against the MPB domain established a clustering of T172, R173, E174, Q196, and T197. Based on the lack of FRET between Trp substitutionally placed at R173 or V198 and bound BZ, this epitope-mapped cluster is located on a separate end of the folded protein from the BZ-binding site. Mutations of the five conserved Cys and Trp residues in the MPB domain gave rise to synergistic and rescuing effects on protein secondary structures and unfolding stability that point to a CCWCW-pentad, reminiscent to the CWC-triad "pin" of immunoglobulin (Ig)-like domains, important for the structural maintenance. These findings, together with secondary structure and fold predictions suggest an anti-parallel beta-strand topology with resemblance to Ig-like fold, having the BZ-binding and the epitopic residues being clustered at two different ends of the fold.

  18. Increased GABA(A receptor ε-subunit expression on ventral respiratory column neurons protects breathing during pregnancy.

    Directory of Open Access Journals (Sweden)

    Keith B Hengen

    Full Text Available GABAergic signaling is essential for proper respiratory function. Potentiation of this signaling with allosteric modulators such as anesthetics, barbiturates, and neurosteroids can lead to respiratory arrest. Paradoxically, pregnant animals continue to breathe normally despite nearly 100-fold increases in circulating neurosteroids. ε subunit-containing GABA(ARs are insensitive to positive allosteric modulation, thus we hypothesized that pregnant rats increase ε subunit-containing GABA(AR expression on brainstem neurons of the ventral respiratory column (VRC. In vivo, pregnancy rendered respiratory motor output insensitive to otherwise lethal doses of pentobarbital, a barbiturate previously used to categorize the ε subunit. Using electrode array recordings in vitro, we demonstrated that putative respiratory neurons of the preBötzinger Complex (preBötC were also rendered insensitive to the effects of pentobarbital during pregnancy, but unit activity in the VRC was rapidly inhibited by the GABA(AR agonist, muscimol. VRC unit activity from virgin and post-partum females was potently inhibited by both pentobarbital and muscimol. Brainstem ε subunit mRNA and protein levels were increased in pregnant rats, and GABA(AR ε subunit expression co-localized with a marker of rhythm generating neurons (neurokinin 1 receptors in the preBötC. These data support the hypothesis that pregnancy renders respiratory motor output and respiratory neuron activity insensitive to barbiturates, most likely via increased ε subunit-containing GABA(AR expression on respiratory rhythm-generating neurons. Increased ε subunit expression may be critical to preserve respiratory function (and life despite increased neurosteroid levels during pregnancy.

  19. Schedule of NMDA receptor subunit expression and functional channel formation in the course of in vitro-induced neurogenesis.

    Science.gov (United States)

    Varju, P; Schlett, K; Eisel, U; Madarász, E

    2001-06-01

    NE-7C2 neuroectodermal cells derived from forebrain vesicles of p53-deficient mouse embryos (E9) produce neurons and astrocytes in vitro if induced by all-trans retinoic acid. The reproducible morphological stages of neurogenesis were correlated with the expression of various NMDA receptor subunits. RT-PCR studies revealed that GluRepsilon1 and GluRepsilon4 subunit mRNAs were transcribed by both non-induced and neuronally differentiated cells. GluRepsilon3 subunit mRNAs were not synthesized by NE-7C2 cells and increased numbers of messages from the GluRepsilon2 gene were detected only after neural network formation. The presence of the GluRzeta1 protein was detected throughout neural induction, whereas retinoic acid-induced neuron formation elevated the amount of exon 21 (C1)- and exon 22 (C2)-containing GluRzeta1 mRNAs and resulted in the appearance of exon 5 (N1)-containing transcripts. NMDA-elicited Ca(2+)-signals were detected only in cells displaying neuronal morphology, but preceding the appearance of synapsin-I immunoreactivity. Our findings demonstrated that, in spite of the presence of subunits necessary for channel formation, functional channels were formed by NE-7C2 cells no sooner than the time of neurite maturation. The data show that the cell line provides a suitable model to analyse the mechanisms involved in NMDA receptor gene expression before the appearance of synaptic communication.

  20. Expression of the Hippocampal NMDA Receptor GluN1 Subunit and Its Splicing Isoforms in Schizophrenia: Postmortem Study

    Czech Academy of Sciences Publication Activity Database

    Vrajová, M.; Šťastný, František; Horáček, J.; Lochman, J.; Šerý, O.; Peková, S.; Klaschka, Jan; Höschl, C.

    2010-01-01

    Roč. 35, č. 7 (2010), s. 994-1002 ISSN 0364-3190 Grant - others:GA MZd(CZ) NR9324 Institutional research plan: CEZ:AV0Z50110509; CEZ:AV0Z10300504 Keywords : schizophrenia * hippocampus * GluN1 subunit of NMDA receptor * splice variants * laterality Subject RIV: FL - Psychiatry, Sexuology Impact factor: 2.608, year: 2010

  1. The N-methyl-D-aspartate receptor subunits NR2A and NR2B bind to the SH2 domains of phospholipase C-gamma.

    Science.gov (United States)

    Gurd, J W; Bissoon, N

    1997-08-01

    The NMDA receptor has recently been found to be phosphorylated on tyrosine. To assess the possible connection between tyrosine phosphorylation of the NMDA receptor and signaling pathways in the postsynaptic cell, we have investigated the relationship between tyrosine phosphorylation and the binding of NMDA receptor subunits to the SH2 domains of phospholipase C-gamma (PLC-gamma). A glutathione S-transferase (GST) fusion protein containing both the N- and the C-proximal SH2 domains of PLC-gamma was bound to glutathione-agarose and reacted with synaptic junctional proteins and glycoproteins. Tyrosine-phosphorylated PSD-GP180, which has been identified as the NR2B subunit of the NMDA receptor, bound to the SH2-agarose beads in a phosphorylation-dependent fashion. Immunoblot analysis with antibodies specific for individual NMDA receptor subunits showed that both NR2A and NR2B subunits bound to the SH2-agarose. No binding occurred to GST-agarose lacking an associated SH2 domain, indicating that binding was specific for the SH2 domains. The binding of receptor subunits increased after the incubation of synaptic junctions with ATP and decreased after treatment of synaptic junctions with exogenous protein tyrosine phosphatase. Immunoprecipitation experiments confirmed that NR2A and NR2B were phosphorylated on tyrosine and further that tyrosine phosphorylation of each of the subunits was increased after incubation with ATP. The results demonstrate that NMDA receptor subunits NR2A and NR2B will bind to the SH2 domains of PLC-gamma and that isolated synaptic junctions contain endogenous protein tyrosine kinase(s) that can phosphorylate both NR2A and NR2B receptor subunits, and suggest that interaction of the tyrosine-phosphorylated NMDA receptor with proteins that contain SH2 domains may serve to link it to signaling pathways in the postsynaptic cell.

  2. Downregulation of GABA[Subscript A] Receptor Protein Subunits a6, ß2, d, e, ?2, ?, and ?2 in Superior Frontal Cortex of Subjects with Autism

    Science.gov (United States)

    Fatemi, S. Hossein; Reutiman, Teri J.; Folsom, Timothy D.; Rustan, Oyvind G.; Rooney, Robert J.; Thuras, Paul D.

    2014-01-01

    We measured protein and mRNA levels for nine gamma-aminobutyric acid A (GABA[subscript A]) receptor subunits in three brain regions (cerebellum, superior frontal cortex, and parietal cortex) in subjects with autism versus matched controls. We observed changes in mRNA for a number of GABA[subscript A] and GABA[subscript B] subunits and overall…

  3. The Drosophila nicotinic acetylcholine receptor subunits Dα5 and Dα7 form functional homomeric and heteromeric ion channels

    Directory of Open Access Journals (Sweden)

    Lansdell Stuart J

    2012-06-01

    Full Text Available Abstract Background Nicotinic acetylcholine receptors (nAChRs play an important role as excitatory neurotransmitters in vertebrate and invertebrate species. In insects, nAChRs are the site of action of commercially important insecticides and, as a consequence, there is considerable interest in examining their functional properties. However, problems have been encountered in the successful functional expression of insect nAChRs, although a number of strategies have been developed in an attempt to overcome such difficulties. Ten nAChR subunits have been identified in the model insect Drosophila melanogaster (Dα1-Dα7 and Dβ1-Dβ3 and a similar number have been identified in other insect species. The focus of the present study is the Dα5, Dα6 and Dα7 subunits, which are distinguished by their sequence similarity to one another and also by their close similarity to the vertebrate α7 nAChR subunit. Results A full-length cDNA clone encoding the Drosophila nAChR Dα5 subunit has been isolated and the properties of Dα5-, Dα6- and Dα7-containing nAChRs examined in a variety of cell expression systems. We have demonstrated the functional expression, as homomeric nAChRs, of the Dα5 and Dα7 subunits in Xenopus oocytes by their co-expression with the molecular chaperone RIC-3. Also, using a similar approach, we have demonstrated the functional expression of a heteromeric ‘triplet’ nAChR (Dα5 + Dα6 + Dα7 with substantially higher apparent affinity for acetylcholine than is seen with other subunit combinations. In addition, specific cell-surface binding of [125I]-α-bungarotoxin was detected in both Drosophila and mammalian cell lines when Dα5 was co-expressed with Dα6 and RIC-3. In contrast, co-expression of additional subunits (including Dα7 with Dα5 and Dα6 prevented specific binding of [125I]-α-bungarotoxin in cell lines, suggesting that co-assembly with other nAChR subunits can block maturation of correctly folded nAChRs in

  4. Ocular myasthenia gravis induced by human acetylcholine receptor ϵ subunit immunization in HLA DR3 transgenic mice.

    Science.gov (United States)

    Wu, Xiaorong; Tuzun, Erdem; Saini, Shamsher S; Wang, Jun; Li, Jing; Aguilera-Aguirre, Leopoldo; Huda, Ruksana; Christadoss, Premkumar

    2015-12-01

    Extraocular muscles (EOM) are preferentially involved in myasthenia gravis (MG) and acetylcholine receptor (AChR) antibody positive MG patients may occasionally present with isolated ocular symptoms. Although experimental autoimmune myasthenia gravis (EAMG) induced by whole AChR immunization closely mimics clinical and immunopathological aspects of MG, EOM are usually not affected. We have previously developed an EAMG model, which imitates EOM symptoms of MG by immunization of human leukocyte antigen (HLA) transgenic mice with α or γ-subunits of human AChR (H-AChR). To investigate the significance of the ϵ-subunit in ocular MG, we immunized HLA-DR3 and HLA-DQ8 transgenic mice with recombinant H-AChR ϵ-subunit expressed in Escherichia coli. HLA-DR3 transgenic mice showed significantly higher clinical ocular and generalized MG severity scores and lower grip strength values than HLA-DQ8 mice. H-AChR ϵ-subunit-immunized HLA-DR3 transgenic mice had higher serum anti-AChR antibody (IgG, IgG1, IgG2b, IgG2c and IgM) levels, neuromuscular junction IgG and complement deposit percentages than ϵ-subunit-immunized HLA-DQ8 transgenic mice. Control mice immunized with E. coli extract or complete Freund adjuvant (CFA) did not show clinical and immunopathological features of ocular and generalized EAMG. Lymph node cells of ϵ-subunit-immunized HLA-DR3 mice showed significantly higher proliferative responses than those of ϵ-subunit-immunized HLA-DQ8 mice, crude E. coli extract-immunized and CFA-immunized transgenic mice. Our results indicate that the human AChR ϵ-subunit is capable of inducing myasthenic muscle weakness. Diversity of the autoimmune responses displayed by mice expressing different HLA class II molecules suggests that the interplay between HLA class II alleles and AChR subunits might have a profound impact on the clinical course of MG. Copyright © 2015 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  5. Adult naked mole-rat brain retains the NMDA receptor subunit GluN2D associated with hypoxia tolerance in neonatal mammals.

    Science.gov (United States)

    Peterson, Bethany L; Park, Thomas J; Larson, John

    2012-01-11

    Adult naked mole-rats show a number of systemic adaptations to a crowded underground habitat that is low in oxygen and high in carbon dioxide. Remarkably, brain slice tissue from adult naked mole-rats also is extremely tolerant to oxygen deprivation as indicated by maintenance of synaptic transmission under hypoxic conditions as well as by a delayed neuronal depolarization during anoxia. These characteristics resemble hypoxia tolerance in brain slices from neonates in a variety of mammal species. An important component of neonatal tolerance to hypoxia involves the subunit composition of NMDA receptors. Neonates have a high proportion of NMDA receptors with GluN2D subunits which are protective because they retard calcium entry into neurons during hypoxic episodes. Therefore, we hypothesized that adult naked mole-rats retain a protective, neonatal-like, NMDA receptor subunit profile. We used immunoblotting to assess age-related changes in NMDA receptor subunits in naked mole-rats and mice. The results show that adult naked mole-rat brain retains a much greater proportion of the hypoxia-protective GluN2D subunit compared to adult mice. However, age-related changes in other subunits (GluN2A and GluN2B) from the neonatal period to adulthood were comparable in mice and naked mole-rats. Hence, adult naked mole-rat brain only retains the neonatal NMDA receptor subunit that is associated with hypoxia tolerance. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  6. GABAA receptor subunit expression changes in the human Alzheimer's disease hippocampus, subiculum, entorhinal cortex and superior temporal gyrus.

    Science.gov (United States)

    Kwakowsky, Andrea; Calvo-Flores Guzmán, Beatriz; Pandya, Madhavi; Turner, Clinton; Waldvogel, Henry J; Faull, Richard L

    2018-02-27

    Gamma-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the central nervous system. GABA type A receptors (GABA A Rs) are severely affected in Alzheimer's disease (AD). However, the distribution and subunit composition of GABA A Rs in the AD brain are not well understood. This is the first comprehensive study to show brain region- and cell layer-specific alterations in the expression of the GABA A R subunits α1-3, α5, β1-3 and γ2 in the human AD hippocampus, entorhinal cortex and superior temporal gyrus (STG). In late-stage AD tissue samples using immunohistochemistry we found significant alteration of all investigated GABA A Rs subunits except for α3 and β1 that were well preserved. The most prominent changes include an increase in GABA A R α1 expression associated with AD in all layers of the CA3 region, in the stratum (str.) granulare and hilus of the dentate gyrus (DG). We found a significant increase in GABA A R α2 expression in the str. oriens of the CA1-3, str. radiatum of the CA2,3 and decrease in the str. pyramidale of the CA1 region in AD cases. In AD there was a significant increase in GABA A R α5 subunit expression in str. pyramidale, str. oriens of the CA1 region and decrease in the STG. We also found a significant decrease in the GABA A R β3 subunit immunoreactivity in the str. oriens of the CA2, str. granulare and str. moleculare of the DG. In conclusion, these findings indicate that the expression of the GABA A R subunits shows brain region- and layer-specific alterations in AD, and these changes could significantly influence and alter GABA A R function in the disease. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  7. Individual stress vulnerability is predicted by short-term memory and AMPA receptor subunit ratio in the hippocampus.

    Science.gov (United States)

    Schmidt, Mathias V; Trümbach, Dietrich; Weber, Peter; Wagner, Klaus; Scharf, Sebastian H; Liebl, Claudia; Datson, Nicole; Namendorf, Christian; Gerlach, Tamara; Kühne, Claudia; Uhr, Manfred; Deussing, Jan M; Wurst, Wolfgang; Binder, Elisabeth B; Holsboer, Florian; Müller, Marianne B

    2010-12-15

    Increased vulnerability to aversive experiences is one of the main risk factors for stress-related psychiatric disorders as major depression. However, the molecular bases of vulnerability, on the one hand, and stress resilience, on the other hand, are still not understood. Increasing clinical and preclinical evidence suggests a central involvement of the glutamatergic system in the pathogenesis of major depression. Using a mouse paradigm, modeling increased stress vulnerability and depression-like symptoms in a genetically diverse outbred strain, and we tested the hypothesis that differences in AMPA receptor function may be linked to individual variations in stress vulnerability. Vulnerable and resilient animals differed significantly in their dorsal hippocampal AMPA receptor expression and AMPA receptor binding. Treatment with an AMPA receptor potentiator during the stress exposure prevented the lasting effects of chronic social stress exposure on physiological, neuroendocrine, and behavioral parameters. In addition, spatial short-term memory, an AMPA receptor-dependent behavior, was found to be predictive of individual stress vulnerability and response to AMPA potentiator treatment. Finally, we provide evidence that genetic variations in the AMPA receptor subunit GluR1 are linked to the vulnerable phenotype. Therefore, we propose genetic variations in the AMPA receptor system to shape individual stress vulnerability. Those individual differences can be predicted by the assessment of short-term memory, thereby opening up the possibility for a specific treatment by enhancing AMPA receptor function.

  8. Mice Lacking the Alpha9 Subunit of the Nicotinic Acetylcholine Receptor Exhibit Deficits in Frequency Difference Limens and Sound Localization

    Directory of Open Access Journals (Sweden)

    Amanda Clause

    2017-06-01

    Full Text Available Sound processing in the cochlea is modulated by cholinergic efferent axons arising from medial olivocochlear neurons in the brainstem. These axons contact outer hair cells in the mature cochlea and inner hair cells during development and activate nicotinic acetylcholine receptors composed of α9 and α10 subunits. The α9 subunit is necessary for mediating the effects of acetylcholine on hair cells as genetic deletion of the α9 subunit results in functional cholinergic de-efferentation of the cochlea. Cholinergic modulation of spontaneous cochlear activity before hearing onset is important for the maturation of central auditory circuits. In α9KO mice, the developmental refinement of inhibitory afferents to the lateral superior olive is disturbed, resulting in decreased tonotopic organization of this sound localization nucleus. In this study, we used behavioral tests to investigate whether the circuit anomalies in α9KO mice correlate with sound localization or sound frequency processing. Using a conditioned lick suppression task to measure sound localization, we found that three out of four α9KO mice showed impaired minimum audible angles. Using a prepulse inhibition of the acoustic startle response paradigm, we found that the ability of α9KO mice to detect sound frequency changes was impaired, whereas their ability to detect sound intensity changes was not. These results demonstrate that cholinergic, nicotinic α9 subunit mediated transmission in the developing cochlear plays an important role in the maturation of hearing.

  9. Detachment strength of human osteoblasts cultured on hydroxyapatite with various surface roughness. Contribution of integrin subunits.

    Science.gov (United States)

    Kokkinos, Petros A; Koutsoukos, Petros G; Deligianni, Despina D

    2012-06-01

    Hydroxyapatite (HA) has been widely used as a bone substitute in dental, maxillofacial and orthopaedic surgery and as osteoconductive bone substitute or precoating of pedicle screws and cages in spine surgery. The aim of the present study was to investigate the osteoblastic adhesion strength on HA substrata with different surface topography and biochemistry (pre-adsorption of fibronectin) after blocking of specific integrin subunits with monoclonal antibodies. Stoichiometric HA was prepared by precipitation followed by ageing and characterized by SEM, EDX, powder XRD, Raman spectroscopy, TGA, and specific surface area analysis. Human bone marrow derived osteoblasts were cultured on HA disc-shaped substrata which were sintered and polished resulting in two surface roughness grades. For attachment evaluation, cells were incubated with monoclonal antibodies and seeded for 2 h on the substrata. Cell detachment strength was determined using a rotating disc device. Cell detachment strength was surface roughness, fibronectin preadsorption and intergin subunit sensitive.

  10. Reduction in mRNA and protein expression of a nicotinic acetylcholine receptor α8 subunit is associated with resistance to imidacloprid in the brown planthopper, Nilaparvata lugens.

    Science.gov (United States)

    Zhang, Yixi; Wang, Xin; Yang, Baojun; Hu, Yuanyuan; Huang, Lixin; Bass, Chris; Liu, Zewen

    2015-11-01

    Target-site resistance is commonly caused by qualitative changes in insecticide target-receptors and few studies have implicated quantitative changes in insecticide targets in resistance. Here we show that resistance to imidacloprid in a selected strain of Nilaparvata lugens is associated with a reduction in expression levels of the nicotinic acetylcholine receptor (nAChR) subunit Nlα8. Synergism bioassays of the selected strain suggested resistance was conferred, in part, by a target-site mechanism. Sequencing of N. lugens nAChR subunit genes identified no mutations associated with resistance, however, a decrease in mRNA and protein levels of Nlα8 was observed during selection. RNA interference knockdown of Nlα8 decreased the sensitivity of N. lugens to imidacloprid, demonstrating that a decrease in Nlα8 expression is sufficient to confer resistance in vivo. Radioligand binding assays revealed that the affinity of the high-affinity imidacloprid-binding site of native nAChRs was reduced by selection, and reducing the amount of Nlα8 cRNA injected into Xenopus oocytes significantly decreased imidacloprid potency on recombinant receptors. Taken together, these results provide strong evidence that a decrease in Nlα8 levels confers resistance to imidacloprid in N. lugens, and thus provides a rare example of target-site resistance associated with a quantitative rather than qualitative change. In insects, target-site mutations often cause high resistance to insecticides, such as neonicotinoids acting on nicotinic acetylcholine receptors (nAChRs). Here we found that a quantitative change in target-protein level, decrease in mRNA and protein levels of Nlα8, contributed importantly to imidacloprid resistance in Nilaparvata lugens. This finding provides a new target-site mechanism of insecticide resistance. © 2015 International Society for Neurochemistry.

  11. Inverse Effects on Gating and Modulation Caused by a Mutation in the M2-M3 Linker of the GABAA Receptor γ SubunitS⃞

    OpenAIRE

    O'Shea, Sean M.; Williams, Carrie A.; Jenkins, Andrew

    2009-01-01

    M2-M3 linkers are receptor subunit domains known to be critical for the normal function of cysteine-loop ligand-gated ion channels. Previous studies of α and β subunits of type “A” GABA receptors suggest that these linkers couple extracellular elements involved in GABA binding to the transmembrane segments that control the opening of the ion channel. To study the importance of the γ subunit M2-M3 linker, we examined the macroscopic and single-channel effects of an engi...

  12. Genetic ablation of the GluK4 kainate receptor subunit causes anxiolytic and antidepressant-like behavior in mice.

    Science.gov (United States)

    Catches, Justin S; Xu, Jian; Contractor, Anis

    2012-03-17

    There is a clear link between dysregulation of glutamatergic signaling and mood disorders. Genetic variants in the glutamate receptor gene GRIK4, which encodes the kainate receptor subunit GluK4, alter the susceptibility for depression, bipolar disorder and schizophrenia. Here we demonstrate that Grik4(-/-) mice have reduced anxiety and an antidepressant-like phenotype. In the elevated zero-maze, a test for anxiety and risk taking behavior, Grik4(-/-) mice spent significantly more time exploring the open areas of the maze. In anxiogenic tests of marble-burying and novelty-induced suppression of feeding, anxiety-like behavior was consistently reduced in knockout animals. In the forced swim test, a test of learned helplessness that is used to determine depression-like behavior, knockout mice demonstrated significantly less immobility suggesting that Grik4 ablation has an antidepressant-like effect. Finally, in the sucrose preference test, a test for anhedonia in rodents, Grik4(-/-) mice demonstrated increased sucrose preference. Expression of the GluK4 receptor subunit in the forebrain is restricted to the CA3 region of the hippocampus and dentate gyrus regions where KARs are known to modulate synaptic plasticity. We tested whether Grik4 ablation had effects on mossy fiber (MF) plasticity and found there to be a significant impairment in LTP likely through a loss of KAR modulation of excitability of the presynaptic MF axons. These studies demonstrate a clear anxiolytic and antidepressant phenotype associated with ablation of Grik4 and a parallel disruption in hippocampal plasticity, providing support for the importance of this receptor subunit in mood disorders. Copyright © 2011 Elsevier B.V. All rights reserved.

  13. Delta Subunit-Containing Gamma-Aminobutyric Acid A Receptor Disinhibits Lateral Amygdala and Facilitates Fear Expression in Mice.

    Science.gov (United States)

    Liu, Zhi-Peng; He, Qing-Hai; Pan, Han-Qing; Xu, Xiao-Bin; Chen, Wen-Bing; He, Ye; Zhou, Jin; Zhang, Wen-Hua; Zhang, Jun-Yu; Ying, Xiao-Ping; Han, Ren-Wen; Li, Bao-Ming; Gao, Tian-Ming; Pan, Bing-Xing

    2017-06-15

    Maintaining gamma-aminobutyric acidergic (GABAergic) inhibition in the amygdala within a physiological range is critical for the appropriate expression of emotions such as fear and anxiety. The synaptic GABA type A receptor (GABA A R) is generally known to mediate the primary component of amygdala inhibition and prevent inappropriate expression of fear. However, little is known about the contribution of the extrasynaptic GABA A R to amygdala inhibition and fear. By using mice expressing green fluorescent protein in interneurons (INs) and lacking the δ subunit-containing GABA A R (GABA A (δ)R), which is exclusively situated in the extrasynaptic membrane, we systematically investigated the role of GABA A (δ)R in regulating inhibition in the lateral amygdala (LA) and fear learning using the combined approaches of immunohistochemistry, electrophysiology, and behavior. In sharp contrast to the established role of synaptic GABA A R in mediating LA inhibition, we found that either pharmacological or physiological recruitment of GABA A (δ)R resulted in the weakening of GABAergic transmission onto projection neurons in LA while leaving the glutamatergic transmission unaltered, suggesting disinhibition by GABA A (δ)R. The disinhibition arose from IN-specific expression of GABA A (δ)R with its activation decreasing the input resistance of local INs and suppressing their activation. Genetic deletion of GABA A (δ)R attenuated its role in suppressing LA INs and disinhibiting LA. Importantly, the GABA A (δ)R facilitated long-term potentiation in sensory afferents to LA and permitted the expression of learned fear. Our findings suggest that GABA A (δ)R serves as a brake rather than a mediator of GABAergic inhibition in LA. The disinhibition by GABA A (δ)R may help to prevent excessive suppression of amygdala activity and thus ensure the expression of emotion. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  14. Delayed internalization and lack of recycling in a beta2-adrenergic receptor fused to the G protein alpha-subunit

    Directory of Open Access Journals (Sweden)

    Floridi Aristide

    2008-10-01

    Full Text Available Abstract Background Chimeric proteins obtained by the fusion of a G protein-coupled receptor (GPCR sequence to the N-terminus of the G protein α-subunit have been extensively used to investigate several aspects of GPCR signalling. Although both the receptor and the G protein generally maintain a fully functional state in such polypeptides, original observations made using a chimera between the β2-adrenergic receptor (β2AR and Gαs indicated that the fusion to the α-subunit resulted in a marked reduction of receptor desensitization and down-regulation. To further investigate this phenomenon, we have compared the rates of internalization and recycling between wild-type and Gαs-fused β2AR. Results The rate of agonist-induced internalization, measured as the disappearance of cell surface immunofluorescence in HEK293 cells permanently expressing N-terminus tagged receptors, was reduced three-fold by receptor-G protein fusion. However, both fused and non-fused receptors translocated to the same endocytic compartment, as determined by dual-label confocal analysis of cells co-expressing both proteins and transferrin co-localization. Receptor recycling, determined as the reversion of surface immunofluorescence following the addition of antagonist to cells that were previously exposed to agonist, markedly differed between wild-type and fused receptors. While most of the internalized β2AR returned rapidly to the plasma membrane, β2AR-Gαs did not recycle, and the observed slow recovery for the fusion protein immunofluorescence was entirely accounted for by protein synthesis. Conclusion The covalent linkage between β2AR and Gαs does not appear to alter the initial endocytic translocation of the two proteins, although there is reduced efficiency. It does, however, completely disrupt the process of receptor and G protein recycling. We conclude that the physical separation between receptor and Gα is not necessary for the transit to early endosomes

  15. Role of NMDA receptor GluN2D subunit in the antidepressant effects of enantiomers of ketamine.

    Science.gov (United States)

    Ide, Soichiro; Ikekubo, Yuiko; Mishina, Masayoshi; Hashimoto, Kenji; Ikeda, Kazutaka

    2017-11-01

    We investigated the rapid and sustained antidepressant effects of enantiomers of ketamine in N-methyl-d-aspartate (NMDA) receptor GluN2D subunit knockout (GluN2D-KO) mice. Intraperitoneal administration of ketamine or its enantiomers 10 min before the tail-suspension test exerted significant antidepressant effects on restraint stress-induced depression in both wildtype and GluN2D-KO mice. The antidepressant effects of (RS)-ketamine and (S)-ketamine were sustained 96 h after the injection in both wildtype and GluN2D-KO mice, but such sustained antidepressant effects of (R)-ketamine were only observed in wildtype mice. These data suggest that the GluN2D subunit is critical for the sustained antidepressant effects of (R)-ketamine. Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  16. Role of NMDA receptor GluN2D subunit in the antidepressant effects of enantiomers of ketamine

    Directory of Open Access Journals (Sweden)

    Soichiro Ide

    2017-11-01

    Full Text Available We investigated the rapid and sustained antidepressant effects of enantiomers of ketamine in N-methyl-d-aspartate (NMDA receptor GluN2D subunit knockout (GluN2D-KO mice. Intraperitoneal administration of ketamine or its enantiomers 10 min before the tail-suspension test exerted significant antidepressant effects on restraint stress-induced depression in both wildtype and GluN2D-KO mice. The antidepressant effects of (RS-ketamine and (S-ketamine were sustained 96 h after the injection in both wildtype and GluN2D-KO mice, but such sustained antidepressant effects of (R-ketamine were only observed in wildtype mice. These data suggest that the GluN2D subunit is critical for the sustained antidepressant effects of (R-ketamine.

  17. IGF-1-dependent subunit communication of the IGF-1 holoreceptor: Interactions between αβ heterodimeric receptor halves

    International Nuclear Information System (INIS)

    Wilden, P.A.; Treadway, J.L.; Morrison, B.D.; Pessin, J.E.

    1989-01-01

    Examination of 125 I-IGF-1 affinity cross-linking and β-subunit autophosphorylation has indicated that IGF-1 induces a covalent association of isolated αβ heterodimeric IGF-1 receptors into an α 2 β 2 heterotetrameric state, in a similar manner to that observed for the insulin receptor. The formation of the α 2 β 2 heterotetrameric IGF-1 receptor complex from the partially purified αβ heterodimers was time dependent with half-maximal formation in approximately 30 min at saturating IGF-1 concentrations. The IGF-1-dependent association of the partially purified αβ heterodimers into an α 2 β 2 heterotetrameric state was specific for the IGF-1 receptors since IGF-1 was unable to stimulate the protein kinase activity of the purified αβ heterodimeric insulin receptor complex. Incubation of the α 2 β 2 heterotetrameric IGF-1 holoreceptor with the specific sulfhydryl agent iodoacetamide (IAN) did not alter 125 I-IGF-1 binding or IGF-1 stimulation of protein kinase activity. However, IAN treatment of the αβ heterodimeric IGF-1 receptors inhibited the IGF-1 dependent covalent formation of the disulfide-linked α 2 β 2 heterotetrameric complex. These data indicate that IGF-1 induces the covalent association of isolated αβ heterodimeric IGF-1 receptor complexes into a disulfide-linked α 2 β 2 heterotetrameric state whereas Mn/MgATP induces a noncovalent association. Therefore, unlike the insulin receptor in which noncovalent association is sufficient for kinase activation, only the covalent assembly of the IGF-1 receptor αβ heterodimers into the α 2 β 2 heterotetrameric holoreceptor complex is associated with ligand-stimulated protein kinase activation

  18. Bisphenol-A rapidly enhanced passive avoidance memory and phosphorylation of NMDA receptor subunits in hippocampus of young rats

    International Nuclear Information System (INIS)

    Xu Xiaohong; Li Tao; Luo Qingqing; Hong Xing; Xie Lingdan; Tian Dong

    2011-01-01

    Bisphenol-A (BPA), an endocrine disruptor, is found to influence development of brain and behaviors in rodents. The previous study indicated that perinatal exposure to BPA impaired learning-memory and inhibited N-methyl-D-aspartate receptor (NMDAR) subunits expressions in hippocampus during the postnatal development in rats; and in cultured hippocampal neurons, BPA rapidly promotes dynamic changes in dendritic morphology through estrogen receptor-mediated pathway by concomitant phosphorylation of NMDAR subunit NR2B. In the present study, we examined the rapid effect of BPA on passive avoidance memory and NMDAR in the developing hippocampus of Sprague-Dawley rats at the age of postnatal day 18. The results showed that BPA or estradiol benzoate (EB) rapidly extended the latency to step down from the platform 1 h after footshock and increased the phosphorylation levels of NR1, NR2B, and mitogen-activated extracellular signal-regulated kinase (ERK) in hippocampus within 1 h. While 24 h after BPA or EB treatment, the improved memory and the increased phosphorylation levels of NR1, NR2B, ERK disappeared. Furthermore, pre-treatment with an estrogen receptors (ERs) antagonist, ICI182,780, or an ERK-activating kinase inhibitor, U0126, significantly attenuated EB- or BPA-induced phosphorylations of NR1, NR2B, and ERK within 1 h. These data suggest that BPA rapidly enhanced short-term passive avoidance memory in the developing rats. A non-genomic effect via ERs may mediate the modulation of the phosphorylation of NMDAR subunits NR1 and NR2B through ERK signaling pathway. - Highlights: → BPA rapidly extended the latency to step down from platform 1 h after footshock. → BPA rapidly increased pNR1, pNR2B, and pERK in hippocampus within 1 h. → ERs antagonist or MEK inhibitor attenuated BPA-induced pNR1, pNR2B, and pERK.

  19. SAHA (Vorinostat Corrects Inhibitory Synaptic Deficits Caused by Missense Epilepsy Mutations to the GABAA Receptor γ2 Subunit

    Directory of Open Access Journals (Sweden)

    Nela Durisic

    2018-03-01

    Full Text Available The GABAA receptor (GABAAR α1 subunit A295D epilepsy mutation reduces the surface expression of α1A295Dβ2γ2 GABAARs via ER-associated protein degradation. Suberanilohydroxamic acid (SAHA, also known as Vorinostat was recently shown to correct the misfolding of α1A295D subunits and thereby enhance the functional surface expression of α1A295Dβ2γ2 GABAARs. Here we investigated whether SAHA can also restore the surface expression of γ2 GABAAR subunits that incorporate epilepsy mutations (N40S, R43Q, P44S, R138G known to reduce surface expression via ER-associated protein degradation. As a control, we also investigated the γ2K289M epilepsy mutation that impairs gating without reducing surface expression. Effects of mutations were evaluated on inhibitory postsynaptic currents (IPSCs mediated by the major synaptic α1β2γ2 GABAAR isoform. Recordings were performed in neuron-HEK293 cell artificial synapses to minimise contamination by GABAARs of undefined subunit composition. Transfection with α1β2γ2N40S, α1β2γ2R43Q, α1β2γ2P44S and α1β2γ2R138G subunits produced IPSCs with decay times slower than those of unmutated α1β2γ2 GABAARs due to the low expression of mutant γ2 subunits and the correspondingly high expression of slow-decaying α1β2 GABAARs. SAHA pre-treatment significantly accelerated the decay time constants of IPSCs consistent with the upregulation of mutant γ2 subunit expression. This increase in surface expression was confirmed by immunohistochemistry. SAHA had no effect on either the IPSC kinetics or surface expression levels of α1β2γ2K289M GABAARs, confirming its specificity for ER-retained mutant γ2 subunits. We also found that α1β2γ2K289M GABAARs and SAHA-treated α1β2γ2R43Q, α1β2γ2P44S and α1β2γ2R138G GABAARs all mediated IPSCs that decayed at significantly faster rates than wild type receptors as temperature was increased from 22 to 40°C. This may help explain why these mutations cause febrile

  20. Electrophysiological evidence for the presence of NR2C subunits of N-methyl-D-aspartate receptors in rat neurons of the nucleus tractus solitarius

    Directory of Open Access Journals (Sweden)

    V. Baptista

    2005-01-01

    Full Text Available The nucleus tractus solitarius (NTS plays an important role in the control of autonomic reflex functions. Glutamate, acting on N-methyl-D-aspartate (NMDA and non-NMDA ionotropic receptors, is the major neurotransmitter in this nucleus, and the relative contribution of each receptor to signal transmission is unclear. We have examined NMDA excitatory postsynaptic currents (NMDA-EPSCs in the subpostremal NTS using the whole cell patch clamp technique on a transverse brainstem slice preparation. The NMDA-EPSCs were evoked by stimulation of the solitary tract over a range of membrane potentials. The NMDA-EPSCs, isolated pharmacologically, presented the characteristic outward rectification and were completely blocked by 50 µM DL-2-amino-5-phosphonopentanoic acid. The I-V relationship of the NMDA response shows that current, with a mean (± SEM amplitude of -41.2 ± 5.5 pA, is present even at a holding potential of -60 mV, suggesting that the NMDA receptors are weakly blocked by extracellular Mg2+ at near resting membrane potentials. This weak block can also be inferred from the value of 0.67 ± 0.17 for parameter delta obtained from a fit of the Woodhull equation to the I-V relationship. The maximal inward current measured on the I-V relationship was at -38.7 ± 4.2 mV. The decay phase of the NMDA currents was fitted with one exponential function with a decay time constant of 239 ± 51 and 418 ± 80 ms at a holding potential of -60 and +50 mV, respectively, which became slower with depolarization (e-fold per 145 mV. The biophysical properties of the NMDA receptors observed in the present study suggest that these receptors in the NTS contain NR2C subunits and may contribute to the synaptic signal integration.

  1. Role of desensitization and subunit expression for kainate receptor-mediated neurotoxicity in murine neocortical cultures

    DEFF Research Database (Denmark)

    Jensen, J B; Schousboe, A; Pickering, D S

    1999-01-01

    ) toxicity mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, and (3) toxicity that can be mediated by kainate receptors when desensitization of the receptors is blocked. The indirect action at NMDA receptors was discovered because (5R, 10S)-(+)-5-methyl-10,11-dihydro-5H...... nedioxy-5H-2,3-benzodiazepine (GYKI 53655), a selective AMPA receptor antagonist, abolished the remaining toxicity. These results indicated that kainate- and domoate-mediated toxicity involves both the NMDA and the AMPA receptors. Pretreatment of the cultures with concanavalin A to prevent desensitization...

  2. The Sleep–Wake Cycle in the Nicotinic Alpha-9 Acetylcholine Receptor Subunit Knock-Out Mice

    Directory of Open Access Journals (Sweden)

    Natalia Madrid-López

    2017-10-01

    Full Text Available There is a neural matrix controlling the sleep–wake cycle (SWC embedded within high ranking integrative mechanisms in the central nervous system. Nicotinic alpha-9 acetylcholine receptor subunit (alpha-9 nAChR participate in physiological processes occurring in sensory, endocrine and immune systems. There is a relationship between the SWC architecture, body homeostasis and sensory afferents so that disruption of afferent signaling is expected to affect the temporal organization of sleep and wake states. The analysis of the SWC of 9 nAChR knock-out animals may help to reveal the contribution of alpha-9 nAChR to sleep chronobiological determinants. Here we explore the polysomnogram in chronically implanted alpha-9 nAChR knock-out (KO and wild-type (WT individuals of the hybrid CBA/Sv129 mouse strain. Records were obtained in isolation chambers under a stable 12:12 light:dark cycle (LD. To unmask the 24-h modulation of the SWC a skeleton photoperiod (SP protocol was performed. Under LD the daily quota (in % of wakefulness (W, NREM sleep and REM sleep obtained in KO and WT animals were 45, 48 and 7, and 46, 46 and 8 respectively. Both groups exhibit nocturnal phase preference of W as well as diurnal and unimodal phase preference of NREM and REM sleep. The acrophase mean angles of KO vs. WT genotypes were not different (Zeitgeber Time: 6.5 vs. 14.9 for W, 4.3 vs. 2.8 for NREM sleep and 5.3 vs. 3.4 for REM sleep, respectively. Transference to SP do not affect daily state quotas, phase preferences and acrophases among genotypes. Unmasking phenomena of the SWC such as wake increment during the rest phase under SP was evident only among WT mice suggesting the involvement of retinal structures containing alpha-9 nAChR in masking processes. Furthermore, KO animals exhibit longer NREM and REM sleep episodes that is independent of illumination conditions. Consolidated diurnal NREM sleep contributed to obtain higher values of NREM sleep delta-EEG activity

  3. The membrane-cytoplasm interface of integrin alpha subunits is critical for receptor latency.

    OpenAIRE

    Briesewitz, R; Kern, A; Smilenov, L B; David, F S; Marcantonio, E E

    1996-01-01

    Localization of integrin receptors to focal contact sites occurs upon ligand binding. This activity is latent, since unoccupied integrin receptors do not localize to focal contacts. Deletion analysis has revealed that the alpha cytoplasmic domains is required for the maintenance of integrin receptor latency. Our current hypothesis for the mechanism of integrin post-ligand binding events is that there is a change in relationship of alpha and beta cytoplasmic domains, which overcomes receptor l...

  4. Molecular basis of the γ-aminobutyric acid A receptor α3 subunit interaction with the clustering protein gephyrin

    DEFF Research Database (Denmark)

    Tretter, Verena; Kerschner, Bernd; Milenkovic, Ivan

    2011-01-01

    The multifunctional scaffolding protein gephyrin is a key player in the formation of the postsynaptic scaffold at inhibitory synapses, clustering both inhibitory glycine receptors (GlyRs) and selected GABA(A) receptor (GABA(A)R) subtypes. We report a direct interaction between the GABA(A)R α3...... subunit and gephyrin, mapping reciprocal binding sites using mutagenesis, overlay, and yeast two-hybrid assays. This analysis reveals that critical determinants of this interaction are located in the motif FNIVGTTYPI in the GABA(A)R α3 M3-M4 domain and the motif SMDKAFITVL at the N terminus...... of the gephyrin E domain. GABA(A)R α3 gephyrin binding-site mutants were unable to co-localize with endogenous gephyrin in transfected hippocampal neurons, despite being able to traffic to the cell membrane and form functional benzodiazepine-responsive GABA(A)Rs in recombinant systems. Interestingly, motifs...

  5. Kalirin Binds the NR2B Subunit of the NMDA Receptor, Altering Its Synaptic Localization and Function

    KAUST Repository

    Kiraly, D. D.

    2011-08-31

    The ability of dendritic spines to change size and shape rapidly is critical in modulating synaptic strength; these morphological changes are dependent upon rearrangements of the actin cytoskeleton. Kalirin-7 (Kal7), a Rho guanine nucleotide exchange factor localized to the postsynaptic density (PSD), modulates dendritic spine morphology in vitro and in vivo. Kal7 activates Rac and interacts with several PSD proteins, including PSD-95, DISC-1, AF-6, and Arf6. Mice genetically lacking Kal7 (Kal7KO) exhibit deficient hippocampal long-term potentiation (LTP) as well as behavioral abnormalities in models of addiction and learning. Purified PSDs from Kal7KO mice contain diminished levels of NR2B, an NMDA receptor subunit that plays a critical role in LTP induction. Here we demonstrate that Kal7KO animals have decreased levels of NR2B-dependent NMDA receptor currents in cortical pyramidal neurons as well as a specific deficit in cell surface expression of NR2B. Additionally, we demonstrate that the genotypic differences in conditioned place preference and passive avoidance learning seen in Kal7KO mice are abrogated when animals are treated with an NR2B-specific antagonist during conditioning. Finally, we identify a stable interaction between the pleckstrin homology domain of Kal7 and the juxtamembrane region of NR2B preceding its cytosolic C-terminal domain. Binding of NR2B to a protein that modulates the actin cytoskeleton is important, as NMDA receptors require actin integrity for synaptic localization and function. These studies demonstrate a novel and functionally important interaction between the NR2B subunit of the NMDA receptor and Kalirin, proteins known to be essential for normal synaptic plasticity.

  6. Kalirin Binds the NR2B Subunit of the NMDA Receptor, Altering Its Synaptic Localization and Function

    KAUST Repository

    Kiraly, D. D.; Lemtiri-Chlieh, Fouad; Levine, E. S.; Mains, R. E.; Eipper, B. A.

    2011-01-01

    The ability of dendritic spines to change size and shape rapidly is critical in modulating synaptic strength; these morphological changes are dependent upon rearrangements of the actin cytoskeleton. Kalirin-7 (Kal7), a Rho guanine nucleotide exchange factor localized to the postsynaptic density (PSD), modulates dendritic spine morphology in vitro and in vivo. Kal7 activates Rac and interacts with several PSD proteins, including PSD-95, DISC-1, AF-6, and Arf6. Mice genetically lacking Kal7 (Kal7KO) exhibit deficient hippocampal long-term potentiation (LTP) as well as behavioral abnormalities in models of addiction and learning. Purified PSDs from Kal7KO mice contain diminished levels of NR2B, an NMDA receptor subunit that plays a critical role in LTP induction. Here we demonstrate that Kal7KO animals have decreased levels of NR2B-dependent NMDA receptor currents in cortical pyramidal neurons as well as a specific deficit in cell surface expression of NR2B. Additionally, we demonstrate that the genotypic differences in conditioned place preference and passive avoidance learning seen in Kal7KO mice are abrogated when animals are treated with an NR2B-specific antagonist during conditioning. Finally, we identify a stable interaction between the pleckstrin homology domain of Kal7 and the juxtamembrane region of NR2B preceding its cytosolic C-terminal domain. Binding of NR2B to a protein that modulates the actin cytoskeleton is important, as NMDA receptors require actin integrity for synaptic localization and function. These studies demonstrate a novel and functionally important interaction between the NR2B subunit of the NMDA receptor and Kalirin, proteins known to be essential for normal synaptic plasticity.

  7. The residence time of GABA(A)Rs at inhibitory synapses is determined by direct binding of the receptor α1 subunit to gephyrin

    DEFF Research Database (Denmark)

    Mukherjee, Jayanta; Kretschmannova, Karla; Gouzer, Geraldine

    2011-01-01

    The majority of fast synaptic inhibition in the brain is mediated by benzodiazepine-sensitive α1-subunit-containing GABA type A receptors (GABA(A)Rs); however, our knowledge of the mechanisms neurons use to regulate their synaptic accumulation is rudimentary. Using immunoprecipitation, we....... Mutating residues 360-375 decreases both the accumulation of α1-containing GABA(A)Rs at gephyrin-positive inhibitory synapses in hippocampal neurons and the amplitude of mIPSCs. We also demonstrate that the affinity of gephyrin for the α1 subunit is modulated by Thr375, a putative phosphorylation site....... Mutation of Thr375 to a phosphomimetic, negatively charged amino acid decreases both the affinity of the α1 subunit for gephyrin, and therefore receptor accumulation at synapses, and the amplitude of mIPSCs. Finally, single-particle tracking reveals that gephyrin reduces the diffusion of α1-subunit...

  8. A reduction in hippocampal GABAA receptor alpha5 subunits disrupts the memory for location of objects in mice.

    Science.gov (United States)

    Prut, L; Prenosil, G; Willadt, S; Vogt, K; Fritschy, J-M; Crestani, F

    2010-07-01

    The memory for location of objects, which binds information about objects to discrete positions or spatial contexts of occurrence, is a form of episodic memory particularly sensitive to hippocampal damage. Its early decline is symptomatic for elderly dementia. Substances that selectively reduce alpha5-GABA(A) receptor function are currently developed as potential cognition enhancers for Alzheimer's syndrome and other dementia, consistent with genetic studies implicating these receptors that are highly expressed in hippocampus in learning performance. Here we explored the consequences of reduced GABA(A)alpha5-subunit contents, as occurring in alpha5(H105R) knock-in mice, on the memory for location of objects. This required the behavioral characterization of alpha5(H105R) and wild-type animals in various tasks examining learning and memory retrieval strategies for objects, locations, contexts and their combinations. In mutants, decreased amounts of alpha5-subunits and retained long-term potentiation in hippocampus were confirmed. They exhibited hyperactivity with conserved circadian rhythm in familiar actimeters, and normal exploration and emotional reactivity in novel places, allocentric spatial guidance, and motor pattern learning acquisition, inhibition and flexibility in T- and eight-arm mazes. Processing of object, position and context memories and object-guided response learning were spared. Genotype difference in object-in-place memory retrieval and in encoding and response learning strategies for object-location combinations manifested as a bias favoring object-based recognition and guidance strategies over spatial processing of objects in the mutants. These findings identify in alpha5(H105R) mice a behavioral-cognitive phenotype affecting basal locomotion and the memory for location of objects indicative of hippocampal dysfunction resulting from moderately decreased alpha5-subunit contents.

  9. Leptin reverses corticosterone-induced inhibition of neural stem cell proliferation through activating the NR2B subunits of NMDA receptors

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Wen-Zhu [Anesthesia and Operation Center, Hainan Branch of Chinese PLA General Hospital, Hainan 572013 (China); Anesthesia and Operation Center, Chinese PLA General Hospital, Beijing 100853 (China); Miao, Yu-Liang [Department of Anesthesiology, PLA No. 306 Hospital, Beijing 100101 (China); Guo, Wen-Zhi [Department of Anesthesiology, Beijing Military General Hospital of Chinese People’s Liberation Army, Beijing 100700 (China); Wu, Wei, E-mail: wwzwgk@163.com [Department of Head and Neck Surgery of Otolaryngology, PLA No. 306 Hospital, Beijing 100101 (China); Li, Bao-Wei [Department of Head and Neck Surgery of Otolaryngology, PLA No. 306 Hospital, Beijing 100101 (China); An, Li-Na [Department of Anesthesiology, Armed Police General Hospital, Beijing 100039 (China); Fang, Wei-Wu [Department of Anesthesiology, PLA No. 306 Hospital, Beijing 100101 (China); Mi, Wei-Dong, E-mail: elite2005gg@163.com [Anesthesia and Operation Center, Chinese PLA General Hospital, Beijing 100853 (China)

    2014-04-25

    Highlights: • Leptin promotes the proliferation of neural stem cells isolated from embryonic mouse hippocampus. • Leptin reverses corticosterone-induced inhibition of neural stem cell proliferation. • The effects of leptin are partially mediated by upregulating NR2B subunits. - Abstract: Corticosterone inhibits the proliferation of hippocampal neural stem cells (NSCs). The removal of corticosterone-induced inhibition of NSCs proliferation has been reported to contribute to neural regeneration. Leptin has been shown to regulate brain development, improve angiogenesis, and promote neural regeneration; however, its effects on corticosterone-induced inhibition of NSCs proliferation remain unclear. Here we reported that leptin significantly promoted the proliferation of hippocampal NSCs in a concentration-dependent pattern. Also, leptin efficiently reversed the inhibition of NSCs proliferation induced by corticosterone. Interestingly, pre-treatment with non-specific NMDA antagonist MK-801, specific NR2B antagonist Ro 25-6981, or small interfering RNA (siRNA) targeting NR2B, significantly blocked the effect of leptin on corticosterone-induced inhibition of NSCs proliferation. Furthermore, corticosterone significantly reduced the protein expression of NR2B, whereas pre-treatment with leptin greatly reversed the attenuation of NR2B expression caused by corticosterone in cultured hippocampal NSCs. Our findings demonstrate that leptin reverses the corticosterone-induced inhibition of NSCs proliferation. This process is, at least partially mediated by increased expression of NR2B subunits of NMDA receptors.

  10. Association of Common Polymorphisms in the Nicotinic Acetylcholine Receptor Alpha4 Subunit Gene with an Electrophysiological Endophenotype in a Large Population-Based Sample.

    Directory of Open Access Journals (Sweden)

    A Mobascher

    Full Text Available Variation in genes coding for nicotinic acetylcholine receptor (nAChR subunits affect cognitive processes and may contribute to the genetic architecture of neuropsychiatric disorders. Single nucleotide polymorphisms (SNPs in the CHRNA4 gene that codes for the alpha4 subunit of alpha4/beta2-containing receptors have previously been implicated in aspects of (mostly visual attention and smoking-related behavioral measures. Here we investigated the effects of six synonymous but functional CHRNA4 exon 5 SNPs on the N100 event-related potential (ERP, an electrophysiological endophenotype elicited by a standard auditory oddball. A total of N = 1,705 subjects randomly selected from the general population were studied with electroencephalography (EEG as part of the German Multicenter Study on nicotine addiction. Two of the six variants, rs1044396 and neighboring rs1044397, were significantly associated with N100 amplitude. This effect was pronounced in females where we also observed an effect on reaction time. Sequencing of the complete exon 5 region in the population sample excluded the existence of additional/functional variants that may be responsible for the observed effects. This is the first large-scale population-based study investigation the effects of CHRNA4 SNPs on brain activity measures related to stimulus processing and attention. Our results provide further evidence that common synonymous CHRNA4 exon 5 SNPs affect cognitive processes and suggest that they also play a role in the auditory system. As N100 amplitude reduction is considered a schizophrenia-related endophenotype the SNPs studied here may also be associated with schizophrenia outcome measures.

  11. Neonatal seizures alter NMDA glutamate receptor GluN2A and 3A subunit expression and function in hippocampal CA1 neurons

    Science.gov (United States)

    Zhou, Chengwen; Sun, Hongyu; Klein, Peter M.; Jensen, Frances E.

    2015-01-01

    Neonatal seizures are commonly caused by hypoxic and/or ischemic injury during birth and can lead to long-term epilepsy and cognitive deficits. In a rodent hypoxic seizure (HS) model, we have previously demonstrated a critical role for seizure-induced enhancement of the AMPA subtype of glutamate receptor (GluA) in epileptogenesis and cognitive consequences, in part due to GluA maturational upregulation of expression. Similarly, as the expression and function of the N-Methyl-D-aspartate (NMDA) subtype of glutamate receptor (GluN) is also developmentally controlled, we examined how early life seizures during the critical period of synaptogenesis could modify GluN development and function. In a postnatal day (P)10 rat model of neonatal seizures, we found that seizures could alter GluN2/3 subunit composition of GluNs and physiological function of synaptic GluNs. In hippocampal slices removed from rats within 48–96 h following seizures, the amplitudes of synaptic GluN-mediated evoked excitatory postsynaptic currents (eEPSCs) were elevated in CA1 pyramidal neurons. Moreover, GluN eEPSCs showed a decreased sensitivity to GluN2B selective antagonists and decreased Mg2+ sensitivity at negative holding potentials, indicating a higher proportion of GluN2A and GluN3A subunit function, respectively. These physiological findings were accompanied by a concurrent increase in GluN2A phosphorylation and GluN3A protein. These results suggest that altered GluN function and expression could potentially contribute to future epileptogenesis following neonatal seizures, and may represent potential therapeutic targets for the blockade of future epileptogenesis in the developing brain. PMID:26441533

  12. Distinct conformational changes in activated agonist-bound and agonist-free glycine receptor subunits

    DEFF Research Database (Denmark)

    Pless, Stephan Alexander; Lynch, Joseph W

    2009-01-01

    Ligand binding to Cys-loop receptors produces either global conformational changes that lead to activation or local conformational changes that do not. We found that the fluorescence of a fluorophore tethered to R271C in the extracellular M2 region of the alpha1 glycine receptor increases during ...

  13. The different effects of over-expressing murine NMDA receptor 2B subunit in the forebrain on conditioned taste aversion.

    Science.gov (United States)

    Li, Shijia; Gu, Yiran; Meng, Bo; Mei, Bing; Li, Fei

    2010-09-10

    The glutamate transmission system and the N-methyl-D-aspartate receptor (NMDA-R), in particular its 2B subunit (NR2B), have been reported to be possibly related to taste memory as a result of treatment with NMDA antagonists and agonists. In order to further study the role of the NR2B subunit in gustation memory, we applied four different taste aversive tasks to observe the behavior of a transgenic mice model in which the NR2B subunit was specifically over-expressed in the forebrain. We found that in both short- and long-term conditioned taste aversion (CTA) experiments, mice with forebrain expression of the NR2B transgene (Tg) showed significantly enhanced CTA 2 days after training. However, both the Tg and the wild-type (Wt) mice shared the same level of aversive memory on the 30th day after training. In both fast and slow extinction experiments, Tg mice maintained a higher CTA memory than that of control mice in most extinction trials. The third experiment, which involved testing the memory for familiar taste, demonstrated that NR2B augmentation had no benefit on the latent inhibition (LI) of CTA. In addition, the last experiment (two-taste LI) showed a suppression of enhanced CTA in Tg mice when the mice were exposed to both novel and familiar tastes. These data suggested that forebrain NR2B over-expression had different effects on gustatory learning and memory. The transgenic animals were only sensitive to novel but not familiar tastes, and up-regulation of NR2B resulted in enhanced CTA function for only a short period of time. 2010 Elsevier B.V. All rights reserved.

  14. The GABAA receptor α and β subunits but not the density of muscimol binding sites are altered in the auditory-linguistic association cortex of subjects with schizophrenia

    International Nuclear Information System (INIS)

    Farnbach-Pralong, D.; Bradbury, R.; Tomaskovic, E.; Copolov, D.; Dean, B.

    1998-01-01

    Full text: An increase in the density of postsynaptic GABA A receptors has recently been reported in the prefrontal cortex of subjects with schizophrenia. This increase has been hypothesised to represent an up-regulation in response a decrease in the density of GABAergic interneurons. In order to determine whether the GABA A receptor is also altered in the auditory-linguistic association cortex of the schizophrenic brain, we used quantitative autoradiography to measure the density of that receptor in tissue obtained at autopsy from 20 control subjects and 20 subjects with schizophrenia matched for sex and age. The density of GABA A receptors was measured as the difference in the binding of the specific ligand [ 3 H]muscimol (100 nM) in the presence or in the absence of 10 5 M SR95531. There was no significant difference in the density of [ 3 H]muscimol binding between tissue from schizophrenic (554.9±20,5 fmol/mg TE) and non-schizophrenic (580.1±26.2 fmol/mg TE) subjects. The abundance of the α and β subunits of the GABA A receptor was also measured in particulate membranes prepared from tissue from 6 control and 6 schizophrenic subjects using Western blots. Detection with monoclonal antibodies and chemiluminescence showed that in tissue from control subjects, there was a significant correlation between the levels of α and β subunits (r=0.817, p=0.047). However, there was no such correlation in tissue from schizophrenic subjects (r=0.265, p=0.61), where in 2 subjects large levels of β-subunit were not matched by similar levels of α subunit. These preliminary results suggest mat there may be a failure for up-regulated GABA A receptor subunits to assemble into functional receptors in this brain region for some subjects with schizophrenia. Copyright (1998) Australian Neuroscience Society

  15. Beta3 subunits promote expression and nicotine-induced up-regulation of human nicotinic alpha6* nicotinic acetylcholine receptors expressed in transfected cell lines.

    Science.gov (United States)

    Tumkosit, Prem; Kuryatov, Alexander; Luo, Jie; Lindstrom, Jon

    2006-10-01

    Nicotinic acetylcholine receptors (AChRs) containing alpha6 subunits are typically found at aminergic nerve endings where they play important roles in nicotine addiction and Parkinson's disease. alpha6* AChRs usually contain beta3 subunits. beta3 subunits are presumed to assemble only in the accessory subunit position within AChRs where they do not participate in forming acetylcholine binding sites. Assembly of subunits in the accessory position may be a critical final step in assembly of mature AChRs. Human alpha6 AChRs subtypes were permanently transfected into human tsA201 human embryonic kidney (HEK) cell lines. alpha6beta2beta3 and alpha6beta4beta3 cell lines were found to express much larger amounts of AChRs and were more sensitive to nicotine-induced increase in the amount of AChRs than were alpha6beta2 or alpha6beta4 cell lines. The increased sensitivity to nicotine-induced up-regulation was due not to a beta3-induced increase in affinity for nicotine but probably to a direct effect on assembly of AChR subunits. HEK cells express only a small amount of mature alpha6beta2 AChRs, but many of these subunits are on the cell surface. This contrasts with Xenopus laevis oocytes, which express a large amount of incorrectly assembled alpha6beta2 subunits that bind cholinergic ligands but form large amorphous intracellular aggregates. Monoclonal antibodies (mAbs) were made to the alpha6 and beta3 subunits to aid in the characterization of these AChRs. The alpha6 mAbs bind to epitopes C-terminal of the extracellular domain. These data demonstrate that both cell type and the accessory subunit beta3 can play important roles in alpha6* AChR expression, stability, and up-regulation by nicotine.

  16. Dopamine Receptor-Specific Contributions to the Computation of Value.

    Science.gov (United States)

    Burke, Christopher J; Soutschek, Alexander; Weber, Susanna; Raja Beharelle, Anjali; Fehr, Ernst; Haker, Helene; Tobler, Philippe N

    2018-05-01

    Dopamine is thought to play a crucial role in value-based decision making. However, the specific contributions of different dopamine receptor subtypes to the computation of subjective value remain unknown. Here we demonstrate how the balance between D1 and D2 dopamine receptor subtypes shapes subjective value computation during risky decision making. We administered the D2 receptor antagonist amisulpride or placebo before participants made choices between risky options. Compared with placebo, D2 receptor blockade resulted in more frequent choice of higher risk and higher expected value options. Using a novel model fitting procedure, we concurrently estimated the three parameters that define individual risk attitude according to an influential theoretical account of risky decision making (prospect theory). This analysis revealed that the observed reduction in risk aversion under amisulpride was driven by increased sensitivity to reward magnitude and decreased distortion of outcome probability, resulting in more linear value coding. Our data suggest that different components that govern individual risk attitude are under dopaminergic control, such that D2 receptor blockade facilitates risk taking and expected value processing.

  17. Long-term potentiation in the CA1 hippocampus induced by NR2A subunit-containing NMDA glutamate receptors is mediated by Ras-GRF2/Erk map kinase signaling.

    Directory of Open Access Journals (Sweden)

    Shan-xue Jin

    Full Text Available BACKGROUND: NMDA-type glutamate receptors (NMDARs are major contributors to long-term potentiation (LTP, a form of synaptic plasticity implicated in the process of learning and memory. These receptors consist of calcium-permeating NR1 and multiple regulatory NR2 subunits. A majority of studies show that both NR2A and NR2B-containing NMDARs can contribute to LTP, but their unique contributions to this form of synaptic plasticity remain poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we show that NR2A and NR2B-containing receptors promote LTP differently in the CA1 hippocampus of 1-month old mice, with the NR2A receptors functioning through Ras-GRF2 and its downstream effector, Erk Map kinase, and NR2B receptors functioning independently of these signaling molecules. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that NR2A-, but not NR2B, containing NMDA receptors induce LTP in pyramidal neurons of the CA1 hippocampus from 1 month old mice through Ras-GRF2 and Erk. This difference add new significance to the observation that the relative levels of these NMDAR subtypes is regulated in neurons, such that NR2A-containing receptors become more prominent late in postnatal development, after sensory experience and synaptic activity.

  18. Regulation of synaptic inhibition by phospho-dependent binding of the AP2 complex to a YECL motif in the GABAA receptor γ2 subunit

    Science.gov (United States)

    Kittler, Josef T.; Chen, Guojun; Kukhtina, Viktoria; Vahedi-Faridi, Ardeschir; Gu, Zhenglin; Tretter, Verena; Smith, Katharine R.; McAinsh, Kristina; Arancibia-Carcamo, I. Lorena; Saenger, Wolfram; Haucke, Volker; Yan, Zhen; Moss, Stephen J.

    2008-01-01

    The regulation of the number of γ2-subunit-containing GABAA receptors (GABAARs) present at synapses is critical for correct synaptic inhibition and animal behavior. This regulation occurs, in part, by the controlled removal of receptors from the membrane in clathrin-coated vesicles, but it remains unclear how clathrin recruitment to surface γ2-subunit-containing GABAARs is regulated. Here, we identify a γ2-subunit-specific Yxxφ-type-binding motif for the clathrin adaptor protein, AP2, which is located within a site for γ2-subunit tyrosine phosphorylation. Blocking GABAAR-AP2 interactions via this motif increases synaptic responses within minutes. Crystallographic and biochemical studies reveal that phosphorylation of the Yxxφ motif inhibits AP2 binding, leading to increased surface receptor number. In addition, the crystal structure provides an explanation for the high affinity of this motif for AP2 and suggests that γ2-subunit-containing heteromeric GABAARs may be internalized as dimers or multimers. These data define a mechanism for tyrosine kinase regulation of GABAAR surface levels and synaptic inhibition. PMID:18305175

  19. Regulation of synaptic inhibition by phospho-dependent binding of the AP2 complex to a YECL motif in the GABAA receptor gamma2 subunit.

    Science.gov (United States)

    Kittler, Josef T; Chen, Guojun; Kukhtina, Viktoria; Vahedi-Faridi, Ardeschir; Gu, Zhenglin; Tretter, Verena; Smith, Katharine R; McAinsh, Kristina; Arancibia-Carcamo, I Lorena; Saenger, Wolfram; Haucke, Volker; Yan, Zhen; Moss, Stephen J

    2008-03-04

    The regulation of the number of gamma2-subunit-containing GABA(A) receptors (GABA(A)Rs) present at synapses is critical for correct synaptic inhibition and animal behavior. This regulation occurs, in part, by the controlled removal of receptors from the membrane in clathrin-coated vesicles, but it remains unclear how clathrin recruitment to surface gamma2-subunit-containing GABA(A)Rs is regulated. Here, we identify a gamma2-subunit-specific Yxxvarphi-type-binding motif for the clathrin adaptor protein, AP2, which is located within a site for gamma2-subunit tyrosine phosphorylation. Blocking GABA(A)R-AP2 interactions via this motif increases synaptic responses within minutes. Crystallographic and biochemical studies reveal that phosphorylation of the Yxxvarphi motif inhibits AP2 binding, leading to increased surface receptor number. In addition, the crystal structure provides an explanation for the high affinity of this motif for AP2 and suggests that gamma2-subunit-containing heteromeric GABA(A)Rs may be internalized as dimers or multimers. These data define a mechanism for tyrosine kinase regulation of GABA(A)R surface levels and synaptic inhibition.

  20. Sex-dependent anti-stress effect of an α5 subunit containing GABAA receptor positive allosteric modulator

    Directory of Open Access Journals (Sweden)

    Sean C. Piantadosi

    2016-11-01

    Full Text Available Rationale: Current first-line treatments for stress-related disorders such as Major Depressive Disorder (MDD act on monoaminergic systems and take weeks to achieve a therapeutic effect with poor response and low remission rates. Recent research has implicated the GABAergic system in the pathophysiology of depression, including deficits in interneurons targeting the dendritic compartment of cortical pyramidal cells. Objectives: The present study evaluates whether SH-053-2'F-R-CH3 (denoted α5-PAM, a positive allosteric modulator selective for α5-subunit containing GABAA receptors found predominantly on cortical pyramidal cell dendrites has anti-stress effects. Methods: Female and male C57BL6/J mice were exposed to unpredictable chronic mild stress (UCMS and treated with α5-PAM acutely (30 minutes prior to assessing behavior or chronically before being assessed behaviorally. Results: Acute and chronic α5-PAM treatments produce a pattern of decreased stress-induced behaviors (denoted as behavioral emotionality across various tests in female, but not in male mice. Behavioral Z-scores calculated across a panel of tests designed to best model the range and heterogeneity of human symptomatology confirmed that acute and chronic α5-PAM treatments consistently produce significant decreases in behavioral emotionality in several independent cohorts of females. The behavioral responses to α5-PAM could not be completely accounted for by differences in drug brain disposition between female and male mice. In mice exposed to UCMS, expression of the Gabra5 gene was increased in the frontal cortex after acute treatment and in hippocampus after chronic treatment with α5-PAM in females only, and these expression changes correlated with behavioral emotionality. Conclusions: We showed that acute and chronic positive modulation of α5 subunit-containing GABAA receptors elicit anti-stress effects in a sex-dependent manner, suggesting novel therapeutic modalities.

  1. Double dissociation of spike timing-dependent potentiation and depression by subunit-preferring NMDA receptor antagonists in mouse barrel cortex.

    Science.gov (United States)

    Banerjee, Abhishek; Meredith, Rhiannon M; Rodríguez-Moreno, Antonio; Mierau, Susanna B; Auberson, Yves P; Paulsen, Ole

    2009-12-01

    Spike timing-dependent plasticity (STDP) is a strong candidate for an N-methyl-D-aspartate (NMDA) receptor-dependent form of synaptic plasticity that could underlie the development of receptive field properties in sensory neocortices. Whilst induction of timing-dependent long-term potentiation (t-LTP) requires postsynaptic NMDA receptors, timing-dependent long-term depression (t-LTD) requires the activation of presynaptic NMDA receptors at layer 4-to-layer 2/3 synapses in barrel cortex. Here we investigated the developmental profile of t-LTD at layer 4-to-layer 2/3 synapses of mouse barrel cortex and studied their NMDA receptor subunit dependence. Timing-dependent LTD emerged in the first postnatal week, was present during the second week and disappeared in the adult, whereas t-LTP persisted in adulthood. An antagonist at GluN2C/D subunit-containing NMDA receptors blocked t-LTD but not t-LTP. Conversely, a GluN2A subunit-preferring antagonist blocked t-LTP but not t-LTD. The GluN2C/D subunit requirement for t-LTD appears to be synapse specific, as GluN2C/D antagonists did not block t-LTD at horizontal cross-columnar layer 2/3-to-layer 2/3 synapses, which was blocked by a GluN2B antagonist instead. These data demonstrate an NMDA receptor subunit-dependent double dissociation of t-LTD and t-LTP mechanisms at layer 4-to-layer 2/3 synapses, and suggest that t-LTD is mediated by distinct molecular mechanisms at different synapses on the same postsynaptic neuron.

  2. Decreased agonist sensitivity of human GABA(A) receptors by an amino acid variant, isoleucine to valine, in the alpha1 subunit.

    Science.gov (United States)

    Westh-Hansen, S E; Rasmussen, P B; Hastrup, S; Nabekura, J; Noguchi, K; Akaike, N; Witt, M R; Nielsen, M

    1997-06-25

    Recombinant human GABA(A) receptors were investigated in vitro by coexpression of cDNAs coding for alpha1, beta2, and gamma2 subunits in the baculovirus/Sf-9 insect cell system. We report that a single amino acid exchange (isoleucine 121 to valine 121) in the N-terminal, extracellular part of the alpha1 subunit induces a marked decrease in agonist GABA(A) receptor ligand sensitivity. The potency of muscimol and GABA to inhibit the binding of the GABA(A) receptor antagonist [3H]SR 95531 (2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl)pyridazinium bromide) was higher in receptor complexes of alpha1(ile 121) beta2gamma2 than in those of alpha1(val 121) beta2gamma2 (IC50 values were 32-fold and 26-fold lower for muscimol and GABA, respectively). The apparent affinity of the GABA(A) receptor antagonist bicuculline methiodide to inhibit the binding of [3H]SR 95531 did not differ between the two receptor complex variants. Electrophysiological measurements of GABA induced whole-cell Cl- currents showed a ten-fold decrease in the GABA(A) receptor sensitivity of alpha1 (val 121) beta2gamma2 as compared to alpha1(ile 121) beta2gamma2 receptor complexes. Thus, a relatively small change in the primary structure of the alpha1 subunit leads to a decrease selective for GABA(A) receptor sensitivity to agonist ligands, since no changes were observed in a GABA(A) receptor antagonist affinity and benzodiazepine receptor binding.

  3. Importance of GluA1 subunit-containing AMPA glutamate receptors for morphine state-dependency.

    Directory of Open Access Journals (Sweden)

    Teemu Aitta-aho

    Full Text Available In state-dependency, information retrieval is most efficient when the animal is in the same state as it was during the information acquisition. State-dependency has been implicated in a variety of learning and memory processes, but its mechanisms remain to be resolved. Here, mice deficient in AMPA-type glutamate receptor GluA1 subunits were first conditioned to morphine (10 or 20 mg/kg s.c. during eight sessions over four days using an unbiased procedure, followed by testing for conditioned place preference at morphine states that were the same as or different from the one the mice were conditioned to. In GluA1 wildtype littermate mice the same-state morphine dose produced the greatest expression of place preference, while in the knockout mice no place preference was then detected. Both wildtype and knockout mice expressed moderate morphine-induced place preference when not at the morphine state (saline treatment at the test; in this case, place preference was weaker than that in the same-state test in wildtype mice. No correlation between place preference scores and locomotor activity during testing was found. Additionally, as compared to the controls, the knockout mice showed unchanged sensitization to morphine, morphine drug discrimination and brain regional μ-opioid receptor signal transduction at the G-protein level. However, the knockout mice failed to show increased AMPA/NMDA receptor current ratios in the ventral tegmental area dopamine neurons of midbrain slices after a single injection of morphine (10 mg/kg, s.c., sliced prepared 24 h afterwards, in contrast to the wildtype mice. The results indicate impaired drug-induced state-dependency in GluA1 knockout mice, correlating with impaired opioid-induced glutamate receptor neuroplasticity.

  4. Cloning of the γ-aminobutyric acid (GABA) ρ1 cDNA: A GABA receptor subunit highly expressed in the retina

    International Nuclear Information System (INIS)

    Cutting, G.R.; Lu, Luo; Kasch, L.M.; Montrose-Rafizadeh, C.; Antonarakis, S.E.; Guggino, W.B.; Kazazian, H.H. Jr.; O'Hara, B.F.; Donovan, D.M.; Shimada, Shoichi; Uhl, G.R.

    1991-01-01

    Type A γ-aminobutyric acid (GABA A ) receptors are a family of ligand-gated chloride channels that are the major inhibitory neurotransmitter receptors in the nervous system. Molecular cloning has revealed diversity in the subunits that compose this heterooligomeric receptor, but each previously elucidated subunit displays amino acid similarity in conserved structural elements. The authors have used these highly conserved regions to identify additional members of this family by using the polymerase chain reaction (PCR). One PCR product was used to isolate a full-length cDNA from a human retina cDNA library. The mature protein predicted from this cDNA sequence is 458 amino acids long and displays between 30 and 38% amino acid similarity to the previously identified GABA A subunits. This gene is expressed primarily in the retina but transcripts are also detected in the brain, lung, and thymus. Injection of Xenopus oocytes with RNA transcribed in vitro produces a GABA-responsive chloride conductance and expression of the cDNA in COS cells yields GABA-displaceable muscimol binding. These features are consistent with our identification of a GABA subunit, GABA ρ 1 , with prominent retinal expression that increases the diversity and tissue specificity of this ligand-gated ion-channel receptor family

  5. Role of the cholinergic nervous system in rheumatoid arthritis: aggravation of arthritis in nicotinic acetylcholine receptor alpha7 subunit gene knockout mice

    NARCIS (Netherlands)

    van Maanen, Marjolein A.; Stoof, Susanne P.; Larosa, Gregory J.; Vervoordeldonk, Margriet J.; Tak, Paul P.

    2010-01-01

    BACKGROUND: The alpha7 subunit of nicotinic acetylcholine receptors (alpha7nAChR) can negatively regulate the synthesis and release of proinflammatory cytokines by macrophages and fibroblast-like synoviocytes in vitro. In addition, stimulation of the alpha7nAChR can reduce the severity of arthritis

  6. Serotonin Transporter (5-HTT) and gamma-Aminobutyric Acid Receptor Subunit beta3 (GABRB3) Gene Polymorphisms are not Associated with Autism in the IMGSA Families

    DEFF Research Database (Denmark)

    Maestrini, E.; Lai, C.; Marlow, A.

    1999-01-01

    Previous studies have suggested that the serotonin transporter (5-HTT) gene and the gamma-aminobutyric acid receptor subunit beta3 (GABRB3) gene, or other genes in the 15q11-q13 region, are possibly involved in susceptibility to autism. To test this hypothesis we performed an association study on...

  7. A juvenile form of postsynaptic hippocampal long-term potentiation in mice deficient for the AMPA receptor subunit GluR-A

    NARCIS (Netherlands)

    Jensen, V.; Kaiser, K.M.M.; Borchardt, T.; Adelmann, G.; Rozov, A.; Burnashev, N.; Brix, C.; Frotscher, M.; Anderson, P.; Hvalby, O.; Sakmann, B.; Seeburg, P.H.; Sprengel, R.

    2003-01-01

    In adult mice, long-term potentiation (LTP) of synaptic transmission at CA3-to-CA1 synapses induced by tetanic stimulation requires L-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors containing GluR-A subunits. Here, we report a GluR-A-independent form of LTP, which is comparable in

  8. Propofol effectively inhibits lithium-pilocarpine- induced status epilepticus in rats via downregulation of N-methyl-D-aspartate receptor 2B subunit expression

    Science.gov (United States)

    Wang, Henglin; Wang, Zhuoqiang; Mi, Weidong; Zhao, Cong; Liu, Yanqin; Wang, Yongan; Sun, Haipeng

    2012-01-01

    Status epilepticus was induced via intraperitoneal injection of lithium-pilocarpine. The inhibitory effects of propofol on status epilepticus in rats were judged based on observation of behavior, electroencephalography and 24-hour survival rate. Propofol (12.5–100 mg/kg) improved status epilepticus in a dose-dependent manner, and significantly reduced the number of deaths within 24 hours of lithium-pilocarpine injection. Western blot results showed that, 24 hours after induction of status epilepticus, the levels of N-methyl-D-aspartate receptor 2A and 2B subunits were significantly increased in rat cerebral cortex and hippocampus. Propofol at 50 mg/kg significantly suppressed the increase in N-methyl-D-aspartate receptor 2B subunit levels, but not the increase in N-methyl-D-aspartate receptor 2A subunit levels. The results suggest that propofol can effectively inhibit status epilepticus induced by lithium-pilocarpine. This effect may be associated with downregulation of N-methyl-D-aspartate receptor 2B subunit expression after seizures. PMID:25737709

  9. Repeated ketamine administration alters N-methyl-d-aspartic acid receptor subunit gene expression: Implication of genetic vulnerability for ketamine abuse and ketamine psychosis in humans

    Science.gov (United States)

    Lipsky, Robert H

    2015-01-01

    For more than 40 years following its approval by the Food and Drug Administration (FDA) as an anesthetic, ketamine, a non-competitive N-methyl-d-aspartic acid (NMDA) receptor antagonist, has been used as a tool of psychiatric research. As a psychedelic drug, ketamine induces psychotic symptoms, cognitive impairment, and mood elevation, which resemble some symptoms of schizophrenia. Recreational use of ketamine has been increasing in recent years. However, little is known of the underlying molecular mechanisms responsible for ketamine-associated psychosis. Recent animal studies have shown that repeated ketamine administration significantly increases NMDA receptor subunit gene expression, in particular subunit 1 (NR1 or GluN1) levels. This results in neurodegeneration, supporting a potential mechanism where up-regulation of NMDA receptors could produce cognitive deficits in chronic ketamine abuse patients. In other studies, NMDA receptor gene variants are associated with addictive behavior. Here, we focus on the roles of NMDA receptor gene subunits in ketamine abuse and ketamine psychosis and propose that full sequencing of NMDA receptor genes may help explain individual vulnerability to ketamine abuse and ketamine-associated psychosis. PMID:25245072

  10. Repeated ketamine administration alters N-methyl-D-aspartic acid receptor subunit gene expression: implication of genetic vulnerability for ketamine abuse and ketamine psychosis in humans.

    Science.gov (United States)

    Xu, Ke; Lipsky, Robert H

    2015-02-01

    For more than 40 years following its approval by the Food and Drug Administration (FDA) as an anesthetic, ketamine, a non-competitive N-methyl-D-aspartic acid (NMDA) receptor antagonist, has been used as a tool of psychiatric research. As a psychedelic drug, ketamine induces psychotic symptoms, cognitive impairment, and mood elevation, which resemble some symptoms of schizophrenia. Recreational use of ketamine has been increasing in recent years. However, little is known of the underlying molecular mechanisms responsible for ketamine-associated psychosis. Recent animal studies have shown that repeated ketamine administration significantly increases NMDA receptor subunit gene expression, in particular subunit 1 (NR1 or GluN1) levels. This results in neurodegeneration, supporting a potential mechanism where up-regulation of NMDA receptors could produce cognitive deficits in chronic ketamine abuse patients. In other studies, NMDA receptor gene variants are associated with addictive behavior. Here, we focus on the roles of NMDA receptor gene subunits in ketamine abuse and ketamine psychosis and propose that full sequencing of NMDA receptor genes may help explain individual vulnerability to ketamine abuse and ketamine-associated psychosis. © 2014 by the Society for Experimental Biology and Medicine.

  11. Cloning and structural analysis of partial acetylcholine receptor subunit genes from the parasitic nematode Teladorsagia circumcincta

    NARCIS (Netherlands)

    Walker, J.; Hoekstra, R.; Roos, M. H.; Wiley, L. J.; Weiss, A. S.; Sangster, N. C.; Tait, A.

    2001-01-01

    Nematode nicotinic acetylcholine receptors (nAChRs) are the sites of action for the anthelmintic drug levamisole. Recent findings indicate that the molecular mechanism of levamisole resistance may involve changes in the number and/or functions of target nAChRs. Accordingly, we have used an RT-PCR

  12. Nicotinic acetylcholine receptor β2-subunits in the medial prefrontal cortex control attention

    NARCIS (Netherlands)

    Guillem, K.; Bloem, B.; Poorthuis, R.B.; Loos, M.; Smit, A.B.; Maskos, U.; Spijker, S.; Mansvelder, H.D.

    2011-01-01

    More than one-third of all people are estimated to experience mild to severe cognitive impairment as they age. Acetylcholine (ACh) levels in the brain diminish with aging, and nicotinic ACh receptor (nAChR) stimulation is known to enhance cognitive performance. The prefrontal cortex (PFC) is

  13. Biochemical and electrophysiological characterization of N-glycans on NMDA receptor subunits

    Czech Academy of Sciences Publication Activity Database

    Kaniaková, Martina; Lichnerová, Katarina; Skřenková, Kristýna; Vyklický ml., Ladislav; Horák, Martin

    2016-01-01

    Roč. 138, č. 4 (2016), s. 546-556 ISSN 0022-3042 R&D Projects: GA ČR(CZ) GA14-02219S Institutional support: RVO:67985823 Keywords : biochemistry * cerebellar granule cells * glutamate receptor * ion channel * patch-clamp Subject RIV: FH - Neurology Impact factor: 4.083, year: 2016

  14. Does the kappa opioid receptor system contribute to pain aversion?

    Directory of Open Access Journals (Sweden)

    Catherine M Cahill

    2014-11-01

    Full Text Available The kappa opioid receptor (KOR and the endogenous peptide-ligand dynorphin have received significant attention due the involvement in mediating a variety of behavioral and neurophysiological responses, including opposing the rewarding properties of drugs of abuse including opioids. Accumulating evidence indicates this system is involved in regulating states of motivation and emotion. Acute activation of the KOR produces an increase in motivational behavior to escape a threat, however, KOR activation associated with chronic stress leads to the expression of symptoms indicative of mood disorders. It is well accepted that KOR can produce analgesia and is engaged in chronic pain states including neuropathic pain. Spinal studies have revealed KOR-induced analgesia in reversing pain hypersensitivities associated with peripheral nerve injury. While systemic administration of KOR agonists attenuates nociceptive sensory transmission, this effect appears to be a stress-induced effect as anxiolytic agents, including delta opioid receptor agonists, mitigate KOR agonist-induced analgesia. Additionally, while the role of KOR and dynorphin in driving the dysphoric and aversive components of stress and drug withdrawal has been well characterized, how this system mediates the negative emotional states associated with chronic pain is relatively unexplored. This review provides evidence that dynorphin and the KOR system contribute to the negative affective component of pain and that this receptor system likely contributes to the high comorbidity of mood disorders associated with chronic neuropathic pain.

  15. Changes in Glutamate/NMDA Receptor Subunit 1 Expression in Rat Brain after Acute and Subacute Exposure to Methamphetamine

    Directory of Open Access Journals (Sweden)

    Walailuk Kerdsan

    2009-01-01

    Full Text Available Methamphetamine (METH is a psychostimulant drug of abuse that produces long-term behavioral changes including behavioral sensitization, tolerance, and dependence. METH has been reported to induce neurotoxic effects in several areas of the brain via the dopaminergic system. Changes of dopamine function can induce malfunction of the glutamatergic system. Therefore, the aim of the present study was to examine the effects of METH administration on the expression of glutamate N-methyl-D-aspartate receptor subunit 1 (NMDAR1 in frontal cortex, striatum, and hippocampal formation after acute and subacute exposure to METH by western blotting. Male Sprague-Dawley rats were injected intraperitoneally with a single dose of 8 mg/kg METH, 4 mg/kg/day METH for 14 days and saline in acute, subacute, and control groups, respectively. A significant increase in NMDAR1 immunoreactive protein was found in frontal cortex in the subacute group (P=.036 but not in the acute group (P=.580. Moreover, a significant increase in NMDAR1 was also observed in striatum in both acute (P=.025 and subacute groups (P=.023. However, no significant differences in NMDAR1 in hippocampal formation were observed in either acute or subacute group. The results suggest that an upregulation of NMDA receptor expression may be a consequence of glutamatergic dysfunction induced by METH.

  16. Investigation of autism and GABA receptor subunit genes in multiple ethnic groups

    OpenAIRE

    Collins, Ann L.; Ma, Deqiong; Whitehead, Patrice L.; Martin, Eden R.; Wright, Harry H.; Abramson, Ruth K.; Hussman, John P.; Haines, Jonathan L.; Cuccaro, Michael L.; Gilbert, John R.; Pericak-Vance, Margaret A.

    2006-01-01

    Autism is a neurodevelopmental disorder of complex genetics, characterized by impairment in social interaction and communication, as well as repetitive behavior. Multiple lines of evidence, including alterations in levels of GABA and GABA receptors in autistic patients, indicate that the GABAergic system, which is responsible for synaptic inhibition in the adult brain, may be involved in autism. Previous studies in our lab indicated association of noncoding single nucleotide polymorphisms (SN...

  17. PKC/CREB pathway mediates the expressions of GABAA receptor subunits in cultured hippocampal neurons after low-Mg2+ solution treatment.

    Science.gov (United States)

    Wu, Guofeng; Yu, Jinpeng; Wang, Likun; Ren, Siying; Zhang, Yixia

    2018-02-01

    To investigate the potential effects of the PKC/CREB pathway on the expressions of GABA A receptor subunits α1, γ2, and δ in cultured hippocampal neurons using a model of epilepsy that employed conditions of low magnesium (Mg 2+ ). A total of 108 embryonic rats at the age of 18 embryonic days (E18)prepared from adult female SD rats were used as experimental subjects. Primary rat hippocampal cultures were prepared from the embryonic 18 days rats. The cultured hippocampal neurons were then treated with artificial cerebrospinal fluid containing low Mg 2+ solutions to generate a low Mg 2+ model of epilepsy. The low Mg 2+ stimulation lasted for 3 h and then returned to in maintenance medium for 20 h. The changes of the GABA A receptor subunit α1, γ2, δ were observed by blocking or activating the function of the CREB. The quantification of the GABA A receptor subunit α1, γ2, δ and the CREB were determined by a qRT-PCR and a Western blot method. After the neurons were exposed to a low-Mg 2+ solution for 3 h, GABA A receptor mRNA expression markedly increased compared to the control, and then gradually decreased. In contrast, CREB mRNA levels exhibited a dramatic down-regulation 3 h after terminating low-Mg 2+ treatment, and then peaked at 9 h. Western blot analyses verified that staurosporine suppressed CREB phosphorylation (p-CREB). The mRNA expression of GABA A receptor subunit α1 increased only in the presence of staurosporine, whereas the expressions of subunits γ2 and δ significantly increased in the presence of either KG-501 or staurosporine. Furthermore, phorbol 12-myristate 13-acetate (PMA) decreased the expressions of GABA A subunits α1, γ2, and δ when administered alone. However, the administration of either KG-501 or staurosporine reversed the inhibitory effects of PMA. The PKC/CREB pathway may negatively regulate the expressions of GABA A receptor subunits α1, γ2, and δ in cultured hippocampal neurons in low Mg 2+ model of

  18. The GABAA Receptor α2 Subunit Activates a Neuronal TLR4 Signal in the Ventral Tegmental Area that Regulates Alcohol and Nicotine Abuse

    Directory of Open Access Journals (Sweden)

    Irina Balan

    2018-04-01

    Full Text Available Alcoholism initiates with episodes of excessive alcohol drinking, known as binge drinking, which is one form of excessive drinking (NIAAA Newsletter, 2004 that is related to impulsivity and anxiety (Ducci et al., 2007; Edenberg et al., 2004 and is also predictive of smoking status. The predisposition of non-alcohol exposed subjects to initiate binge drinking is controlled by neuroimmune signaling that includes an innately activated neuronal Toll-like receptor 4 (TLR4 signal. This signal also regulates cognitive impulsivity, a heritable trait that defines drug abuse initiation. However, the mechanism of signal activation, its function in dopaminergic (TH+ neurons within the reward circuitry implicated in drug-seeking behavior [viz. the ventral tegmental area (VTA], and its contribution to nicotine co-abuse are still poorly understood. We report that the γ-aminobutyric acidA receptor (GABAAR α2 subunit activates the TLR4 signal in neurons, culminating in the activation (phosphorylation/nuclear translocation of cyclic AMP response element binding (CREB but not NF-kB transcription factors and the upregulation of corticotropin-releasing factor (CRF and tyrosine hydroxylase (TH. The signal is activated through α2/TLR4 interaction, as evidenced by co-immunoprecipitation, and it is present in the VTA from drug-untreated alcohol-preferring P rats. VTA infusion of neurotropic herpes simplex virus (HSV vectors for α2 (pHSVsiLA2 or TLR4 (pHSVsiTLR4 but not scrambled (pHSVsiNC siRNA inhibits signal activation and both binge alcohol drinking and nicotine sensitization, suggesting that the α2-activated TLR4 signal contributes to the regulation of both alcohol and nicotine abuse.

  19. Domain cooperativity in the β1a subunit is essential for dihydropyridine receptor voltage sensing in skeletal muscle.

    Science.gov (United States)

    Dayal, Anamika; Bhat, Vinayakumar; Franzini-Armstrong, Clara; Grabner, Manfred

    2013-04-30

    The dihydropyridine receptor (DHPR) β1a subunit is crucial for enhancement of DHPR triad expression, assembly of DHPRs in tetrads, and elicitation of DHPRα1S charge movement--the three prerequisites of skeletal muscle excitation-contraction coupling. Despite the ability to fully target α1S into triadic junctions and tetradic arrays, the neuronal isoform β3 was unable to restore considerable charge movement (measure of α1S voltage sensing) upon expression in β1-null zebrafish relaxed myotubes, unlike the other three vertebrate β-isoforms (β1a, β2a, and β4). Thus, we used β3 for chimerization with β1a to investigate whether any of the five distinct molecular regions of β1a is dominantly involved in inducing the voltage-sensing function of α1S. Surprisingly, systematic domain swapping between β1a and β3 revealed a pivotal role of the src homology 3 (SH3) domain and C terminus of β1a in charge movement restoration. More interestingly, β1a SH3 domain and C terminus, when simultaneously engineered into β3 sequence background, were able to fully restore charge movement together with proper intracellular Ca(2+) release, suggesting cooperativity of these two domains in induction of the α1S voltage-sensing function in skeletal muscle excitation-contraction coupling. Furthermore, substitution of a proline by alanine in the putative SH3-binding polyproline motif in the proximal C terminus of β1a (also of β2a and β4) fully obstructed α1S charge movement. Consequently, we postulate a model according to which β subunits, probably via the SH3-C-terminal polyproline interaction, adapt a discrete conformation required to modify the α1S conformation apt for voltage sensing in skeletal muscle.

  20. The morphological and chemical characteristics of striatal neurons immunoreactive for the alpha1-subunit of the GABA(A) receptor in the rat.

    Science.gov (United States)

    Waldvogel, H J; Kubota, Y; Trevallyan, S C; Kawaguchi, Y; Fritschy, J M; Mohler, H; Faull, R L

    1997-10-01

    The distribution, morphology and chemical characteristics of neurons immunoreactive for the alpha1-subunit of the GABA(A) receptor in the striatum of the basal ganglia in the rat brain were investigated at the light, confocal and electron microscope levels using single, double and triple immunohistochemical labelling techniques. The results showed that alpha1-subunit immunoreactive neurons were sparsely distributed throughout the rat striatum. Double and triple labelling results showed that all the alpha1-subunit-immunoreactive neurons were positive for glutamate decarboxylase and immunoreactive for the beta2,3 and gamma2 subunits of the GABA(A) receptor. Three types of alpha1-subunit-immunoreactive neurons were identified in the striatum on the basis of cellular morphology and chemical characteristics. The most numerous alpha1-subunit-immunoreactive neurons were medium-sized, aspiny neurons with a widely branching dendritic tree. They were parvalbumin-negative and were located mainly in the dorsolateral regions of the striatum. Electron microscopy showed that these neurons had an indented nuclear membrane, typical of striatal interneurons, and were surrounded by small numbers of axon terminals which established alpha1-subunit-immunoreactive synaptic contacts with the soma and dendrites. These cells were classified as type 1 alpha1-subunit-immunoreactive neurons and comprised 75% of the total population of alpha1-subunit-immunoreactive neurons in the striatum. The remaining alpha1-subunit-immunoreactive neurons comprised of a heterogeneous population of large-sized neurons localized in the ventral and medial regions of the striatum. The most numerous large-sized cells were parvalbumin-negative, had two to three relatively short branching dendrites and were designated type 2 alpha1-subunit-immunoreactive neurons. Electron microscopy showed that the type 2 neurons were characterized by a highly convoluted nuclear membrane and were sparsely covered with small axon

  1. Ectopic Expression of α6 and δ GABAA Receptor Subunits in Hilar Somatostatin Neurons Increases Tonic Inhibition and Alters Network Activity in the Dentate Gyrus

    Science.gov (United States)

    Tong, Xiaoping; Peng, Zechun; Zhang, Nianhui; Cetina, Yliana; Huang, Christine S.; Wallner, Martin; Otis, Thomas S.

    2015-01-01

    The role of GABAA receptor (GABAAR)-mediated tonic inhibition in interneurons remains unclear and may vary among subgroups. Somatostatin (SOM) interneurons in the hilus of the dentate gyrus show negligible expression of nonsynaptic GABAAR subunits and very low tonic inhibition. To determine the effects of ectopic expression of tonic GABAAR subtypes in these neurons, Cre-dependent viral vectors were used to express GFP-tagged GABAAR subunits (α6 and δ) selectively in hilar SOM neurons in SOM-Cre mice. In single-transfected animals, immunohistochemistry demonstrated strong expression of either the α6 or δ subunit; in cotransfected animals, both subunits were consistently expressed in the same neurons. Electrophysiology revealed a robust increase of tonic current, with progressively larger increases following transfection of δ, α6, and α6/δ subunits, respectively, indicating formation of functional receptors in all conditions and likely coassembly of the subunits in the same receptor following cotransfection. An in vitro model of repetitive bursting was used to determine the effects of increased tonic inhibition in hilar SOM interneurons on circuit activity in the dentate gyrus. Upon cotransfection, the frequency of GABAAR-mediated bursting in granule cells was reduced, consistent with a reduction in synchronous firing among hilar SOM interneurons. Moreover, in vivo studies of Fos expression demonstrated reduced activation of α6/δ-cotransfected neurons following acute seizure induction by pentylenetetrazole. The findings demonstrate that increasing tonic inhibition in hilar SOM interneurons can alter dentate gyrus circuit activity during strong stimulation and suggest that tonic inhibition of interneurons could play a role in regulating excessive synchrony within the network. SIGNIFICANCE STATEMENT In contrast to many hippocampal interneurons, somatostatin (SOM) neurons in the hilus of the dentate gyrus have very low levels of nonsynaptic GABAARs and exhibit

  2. Contribution of the major and minor subunits to fimbria-mediated adherence of Haemophilus influenzae to human epithelial cells and erythrocytes

    NARCIS (Netherlands)

    van Ham, S. M.; van Alphen, L.; Mooi, F. R.; van Putten, J. P.

    1995-01-01

    Fimbriae are colonization factors of the human pathogen Haemophilus influenzae in that they mediate bacterial adherence to human eukaryotic cells. The contribution of the major (HifA) and putative minor (HifD and HifE) subunits of H. influenzae fimbriae to fimbria-specific adherence was studied by

  3. Comparison of cDNA-derived protein sequences of the human fibronectin and vitronectin receptor α-subunits and platelet glycoprotein IIb

    International Nuclear Information System (INIS)

    Fitzgerald, L.A.; Poncz, M.; Steiner, B.; Rall, S.C. Jr.; Bennett, J.S.; Phillips, D.R.

    1987-01-01

    The fibronectin receptor (FnR), the vitronectin receptor (VnR), and the platelet membrane glycoprotein (GP) IIb-IIIa complex are members of a family of cell adhesion receptors, which consist of noncovalently associated α- and β-subunits. The present study was designed to compare the cDNA-derived protein sequences of the α-subunits of human FnR, VnR, and platelet GP IIb. cDNA clones for the α-subunit of the FnR (FnR/sub α/) were obtained from a human umbilical vein endothelial (HUVE) cell library by using an oligonucleotide probe designed from a peptide sequence of platelet GP IIb. cDNA clones for platelet GP IIb were isolated from a cDNA expression library of human erythroleukemia cells by using antibodies. cDNA clones of the VnR α-subunit (VnR/sub α/) were obtained from the HUVE cell library by using an oligonucleotide probe from the partial cDNA sequence for the VnR/sub α/. Translation of these sequences showed that the FNR/sub α/, the VnR/sub α/, and GP IIb are composed of disulfide-linked large (858-871 amino acids) and small (137-158 amino acids) chains that are posttranslationally processed from a single mRNA. A single hydrophobic segment located near the carboxyl terminus of each small chain appears to be a transmembrane domain. The large chains appear to be entirely extracellular, and each contains four repeated putative Ca 2+ -binding domains of about 30 amino acids that have sequence similarities to other Ca 2+ -binding proteins. The identity among the protein sequences of the three receptor α-subunits ranges from 36.1% to 44.5%, with the Ca 2+ -binding domains having the greatest homology. These proteins apparently evolved by a process of gene duplication

  4. Epac Signaling Is Required for Cocaine-Induced Change in AMPA Receptor Subunit Composition in the Ventral Tegmental Area.

    Science.gov (United States)

    Liu, Xiaojie; Chen, Yao; Tong, Jiaqing; Reynolds, Ashley M; Proudfoot, Sarah C; Qi, Jinshun; Penzes, Peter; Lu, Youming; Liu, Qing-Song

    2016-04-27

    Exchange protein directly activated by cAMP (Epac) and protein kinase A (PKA) are intracellular receptors for cAMP. Although PKA and its downstream effectors have been studied extensively in the context of drug addiction, whether and how Epac regulates cellular and behavioral effects of drugs of abuse remain essentially unknown. Epac is known to regulate AMPA receptor (AMPAR) trafficking. Previous studies have shown that a single cocaine exposure in vivo leads to an increase in GluA2-lacking AMPARs in dopamine neurons of the ventral tegmental area (VTA). We tested the hypothesis that Epac mediates cocaine-induced changes in AMPAR subunit composition in the VTA. We report that a single cocaine injection in vivo in wild-type mice leads to inward rectification of EPSCs and renders EPSCs sensitive to a GluA2-lacking AMPAR blocker in VTA dopamine neurons. The cocaine-induced increase in GluA2-lacking AMPARs was absent in Epac2-deficient mice but not in Epac1-deficient mice. In addition, activation of Epac with the selective Epac agonist 8-CPT-2Me-cAMP (8-CPT) recapitulated the cocaine-induced increase in GluA2-lacking AMPARs, and the effects of 8-CPT were mediated by Epac2. We also show that conditioned place preference to cocaine was impaired in Epac2-deficient mice and in mice in which Epac2 was knocked down in the VTA but was not significantly altered in Epac1-deficient mice. Together, these results suggest that Epac2 is critically involved in the cocaine-induced change in AMPAR subunit composition and drug-cue associative learning. Addictive drugs, such as cocaine, induce long-lasting adaptions in the reward circuits of the brain. A single intraperitoneal injection of cocaine leads to changes in the composition and property of the AMPAR that carries excitatory inputs to dopamine neurons. Here, we provide evidence that exchange protein directly activated by cAMP (Epac), a cAMP sensor protein, is required for the cocaine-induced changes of the AMPAR. We found that the

  5. Mutation of a nicotinic acetylcholine receptor β subunit is associated with resistance to neonicotinoid insecticides in the aphid Myzus persicae

    Directory of Open Access Journals (Sweden)

    Field Linda M

    2011-05-01

    Full Text Available Abstract Background Myzus persicae is a globally important aphid pest with a history of developing resistance to insecticides. Unusually, neonicotinoids have remained highly effective as control agents despite nearly two decades of steadily increasing use. In this study, a clone of M. persicae collected from southern France was found, for the first time, to exhibit sufficiently strong resistance to result in loss of the field effectiveness of neonicotinoids. Results Bioassays, metabolism and gene expression studies implied the presence of two resistance mechanisms in the resistant clone, one based on enhanced detoxification by cytochrome P450 monooxygenases, and another unaffected by a synergist that inhibits detoxifying enzymes. Binding of radiolabeled imidacloprid (a neonicotinoid to whole body membrane preparations showed that the high affinity [3H]-imidacloprid binding site present in susceptible M. persicae is lost in the resistant clone and the remaining lower affinity site is altered compared to susceptible clones. This confers a significant overall reduction in binding affinity to the neonicotinoid target: the nicotinic acetylcholine receptor (nAChR. Comparison of the nucleotide sequence of six nAChR subunit (Mpα1-5 and Mpβ1 genes from resistant and susceptible aphid clones revealed a single point mutation in the loop D region of the nAChR β1 subunit of the resistant clone, causing an arginine to threonine substitution (R81T. Conclusion Previous studies have shown that the amino acid at this position within loop D is a key determinant of neonicotinoid binding to nAChRs and this amino acid change confers a vertebrate-like character to the insect nAChR receptor and results in reduced sensitivity to neonicotinoids. The discovery of the mutation at this position and its association with the reduced affinity of the nAChR for imidacloprid is the first example of field-evolved target-site resistance to neonicotinoid insecticides and also

  6. Selective Attention to Visual Stimuli Using Auditory Distractors Is Altered in Alpha-9 Nicotinic Receptor Subunit Knock-Out Mice.

    Science.gov (United States)

    Terreros, Gonzalo; Jorratt, Pascal; Aedo, Cristian; Elgoyhen, Ana Belén; Delano, Paul H

    2016-07-06

    During selective attention, subjects voluntarily focus their cognitive resources on a specific stimulus while ignoring others. Top-down filtering of peripheral sensory responses by higher structures of the brain has been proposed as one of the mechanisms responsible for selective attention. A prerequisite to accomplish top-down modulation of the activity of peripheral structures is the presence of corticofugal pathways. The mammalian auditory efferent system is a unique neural network that originates in the auditory cortex and projects to the cochlear receptor through the olivocochlear bundle, and it has been proposed to function as a top-down filter of peripheral auditory responses during attention to cross-modal stimuli. However, to date, there is no conclusive evidence of the involvement of olivocochlear neurons in selective attention paradigms. Here, we trained wild-type and α-9 nicotinic receptor subunit knock-out (KO) mice, which lack cholinergic transmission between medial olivocochlear neurons and outer hair cells, in a two-choice visual discrimination task and studied the behavioral consequences of adding different types of auditory distractors. In addition, we evaluated the effects of contralateral noise on auditory nerve responses as a measure of the individual strength of the olivocochlear reflex. We demonstrate that KO mice have a reduced olivocochlear reflex strength and perform poorly in a visual selective attention paradigm. These results confirm that an intact medial olivocochlear transmission aids in ignoring auditory distraction during selective attention to visual stimuli. The auditory efferent system is a neural network that originates in the auditory cortex and projects to the cochlear receptor through the olivocochlear system. It has been proposed to function as a top-down filter of peripheral auditory responses during attention to cross-modal stimuli. However, to date, there is no conclusive evidence of the involvement of olivocochlear

  7. Inter-subunit interactions across the upper voltage sensing-pore domain interface contribute to the concerted pore opening transition of Kv channels.

    Directory of Open Access Journals (Sweden)

    Tzilhav Shem-Ad

    Full Text Available The tight electro-mechanical coupling between the voltage-sensing and pore domains of Kv channels lies at the heart of their fundamental roles in electrical signaling. Structural data have identified two voltage sensor pore inter-domain interaction surfaces, thus providing a framework to explain the molecular basis for the tight coupling of these domains. While the contribution of the intra-subunit lower domain interface to the electro-mechanical coupling that underlies channel opening is relatively well understood, the contribution of the inter-subunit upper interface to channel gating is not yet clear. Relying on energy perturbation and thermodynamic coupling analyses of tandem-dimeric Shaker Kv channels, we show that mutation of upper interface residues from both sides of the voltage sensor-pore domain interface stabilizes the closed channel state. These mutations, however, do not affect slow inactivation gating. We, moreover, find that upper interface residues form a network of state-dependent interactions that stabilize the open channel state. Finally, we note that the observed residue interaction network does not change during slow inactivation gating. The upper voltage sensing-pore interaction surface thus only undergoes conformational rearrangements during channel activation gating. We suggest that inter-subunit interactions across the upper domain interface mediate allosteric communication between channel subunits that contributes to the concerted nature of the late pore opening transition of Kv channels.

  8. Inter-subunit interactions across the upper voltage sensing-pore domain interface contribute to the concerted pore opening transition of Kv channels.

    Science.gov (United States)

    Shem-Ad, Tzilhav; Irit, Orr; Yifrach, Ofer

    2013-01-01

    The tight electro-mechanical coupling between the voltage-sensing and pore domains of Kv channels lies at the heart of their fundamental roles in electrical signaling. Structural data have identified two voltage sensor pore inter-domain interaction surfaces, thus providing a framework to explain the molecular basis for the tight coupling of these domains. While the contribution of the intra-subunit lower domain interface to the electro-mechanical coupling that underlies channel opening is relatively well understood, the contribution of the inter-subunit upper interface to channel gating is not yet clear. Relying on energy perturbation and thermodynamic coupling analyses of tandem-dimeric Shaker Kv channels, we show that mutation of upper interface residues from both sides of the voltage sensor-pore domain interface stabilizes the closed channel state. These mutations, however, do not affect slow inactivation gating. We, moreover, find that upper interface residues form a network of state-dependent interactions that stabilize the open channel state. Finally, we note that the observed residue interaction network does not change during slow inactivation gating. The upper voltage sensing-pore interaction surface thus only undergoes conformational rearrangements during channel activation gating. We suggest that inter-subunit interactions across the upper domain interface mediate allosteric communication between channel subunits that contributes to the concerted nature of the late pore opening transition of Kv channels.

  9. NMDA receptor GluN2A/GluN2B subunit ratio as synaptic trait of levodopa-induced dyskinesias: from experimental models to patients

    Directory of Open Access Journals (Sweden)

    Manuela eMellone

    2015-07-01

    Full Text Available Levodopa-induced dyskinesias (LIDs are major complications in the pharmacological management of Parkinson’s disease (PD. Abnormal glutamatergic transmission in the striatum is considered a key factor in the development of LIDs. This work aims at i. characterizing NMDA receptor GluN2A/GluN2B subunit ratio as a common synaptic trait in rat and primate models of LIDs and in dyskinetic PD patients, and ii. validating the potential therapeutic effect of a cell-permeable peptide interfering with GluN2A synaptic localization on the dyskinetic behavior of these experimental models of LIDs. Here we demonstrate an altered ratio of synaptic GluN2A/GluN2B-containing NMDA receptors in the striatum of levodopa-treated dyskinetic rats and monkeys as well as in post-mortem tissue from dyskinetic PD patients. The modulation of synaptic NMDA receptor composition by a cell-permeable peptide interfering with GluN2A subunit interaction with the scaffolding protein PSD-95 leads to a reduction in the dyskinetic motor behavior in the two animal models of LIDs. Our results indicate that targeting synaptic NMDA receptor subunit composition may represent an intriguing therapeutic approach aimed at ameliorating levodopa motor side effects.

  10. Association between the GABA(A) receptor alpha5 subunit gene locus (GABRA5) and bipolar affective disorder.

    Science.gov (United States)

    Papadimitriou, G N; Dikeos, D G; Karadima, G; Avramopoulos, D; Daskalopoulou, E G; Vassilopoulos, D; Stefanis, C N

    1998-02-07

    Genetic factors seem to play an important role in the pathogenesis of affective disorder. The candidate gene strategies are being used, among others, to identify the genes conferring vulnerability to the disease. The genes coding for the receptors of gamma-aminobutyric acid (GABA) have been proposed as candidates for affective disorder, since the GABA neurotransmitter system has been implicated in the pathogenesis of the illness. We examined the possible genetic association between the GABA(A) receptor alpha5 subunit gene locus (GABRA5) on chromosome 15 and affective disorder, in 48 bipolar patients (BP), 40 unipolar patients (UP), and 50 healthy individuals, age- and sex-matched to the patients. All patients and controls were unrelated Greeks. Diagnoses were made after direct interviews according to the DSM-IV and ICD-10 criteria. For the genotyping, a dinucleotide (CA) repeat marker was used. The polymerase chain reaction (PCR) products found were nine alleles with lengths between 272 and 290 base pairs (bp). The distribution of allelic frequencies of the GABRA5 locus differed significantly between BP patients and controls with the 282-bp allele found to be associated with BP affective disorder, while no such difference was observed between the groups of UP patients and controls nor between the two patient groups. The presence or absence of the 282-bp allele in the genotype of BP patients was not shown to influence the age of onset and the overall clinical severity, but was found to be associated with a preponderance of manic over depressive episodes in the course of the illness.

  11. Distinct α subunit variations of the hypothalamic GABAA receptor triplets (αβγ are linked to hibernating state in hamsters

    Directory of Open Access Journals (Sweden)

    Alò Raffaella

    2010-09-01

    Full Text Available Abstract Background The structural arrangement of the γ-aminobutyric acid type A receptor (GABAAR is known to be crucial for the maintenance of cerebral-dependent homeostatic mechanisms during the promotion of highly adaptive neurophysiological events of the permissive hibernating rodent, i.e the Syrian golden hamster. In this study, in vitro quantitative autoradiography and in situ hybridization were assessed in major hypothalamic nuclei. Reverse Transcription Reaction-Polymerase chain reaction (RT-PCR tests were performed for specific GABAAR receptor subunit gene primers synthases of non-hibernating (NHIB and hibernating (HIB hamsters. Attempts were made to identify the type of αβγ subunit combinations operating during the switching ON/OFF of neuronal activities in some hypothalamic nuclei of hibernators. Results Both autoradiography and molecular analysis supplied distinct expression patterns of all α subunits considered as shown by a strong (p 1 ratio (over total α subunits considered in the present study in the medial preoptic area (MPOA and arcuate nucleus (Arc of NHIBs with respect to HIBs. At the same time α2 subunit levels proved to be typical of periventricular nucleus (Pe and Arc of HIB, while strong α4 expression levels were detected during awakening state in the key circadian hypothalamic station, i.e. the suprachiasmatic nucleus (Sch; 60%. Regarding the other two subunits (β and γ, elevated β3 and γ3 mRNAs levels mostly characterized MPOA of HIBs, while prevalently elevated expression concentrations of the same subunits were also typical of Sch, even though this time during the awakening state. In the case of Arc, notably elevated levels were obtained for β3 and γ2 during hibernating conditions. Conclusion We conclude that different αβγ subunits are operating as major elements either at the onset of torpor or during induction of the arousal state in the Syrian golden hamster. The identification of a brain regional

  12. Association between GABA-A receptor alpha 5 subunit gene locus and schizophrenia of a later age of onset.

    Science.gov (United States)

    Papadimitriou, G; Dikeos, D; Daskalopoulou, E; Karadima, G; Avramopoulos, D; Contis, C; Stefanis, C

    2001-01-01

    Heritability is considered to be a major etiologic factor for schizophrenia. Among the genes considered as candidates for the disease, are those related to GABAergic neurotransmission. Our aim was to test for a genetic association between GABA-A receptor alpha 5 subunit gene locus (GABRA(5)) and schizophrenia. Genotyping of the GABRA(5) locus was performed by the use of a dinucleotide (CA) repeat marker in 46 schizophrenic patients and 50 healthy individuals, all unrelated Greeks. Eight alleles were identified, 276-290 bp long. A nonsignificant excess of the 282-bp allele, which was found in a previous study in a Greek population to be associated with bipolar affective disorder, was observed in schizophrenic patients (33.8 vs. 23.9% in the controls). The frequency of this allele was 43.3% among patients with a later age of onset (over 25 years), differing at a statistically significant level from the controls (p < 0.05). These results suggest that common pathophysiological mechanisms may possibly underlie affective disorders and schizophrenia, at least in a subgroup of patients. Copyright 2001 S. Karger AG, Basel

  13. Mouse hippocampal GABAB1 but not GABAB2 subunit-containing receptor complex levels are paralleling retrieval in the multiple-T-maze

    Directory of Open Access Journals (Sweden)

    Soheil eKeihan Falsafi

    2015-10-01

    Full Text Available GABAB receptors are heterodimeric G-protein coupled receptors known to be involved in learning and memory. Although a role for GABAB receptors in cognitive processes is evident, there is no information on hippocampal GABAB receptor complexes in a multiple T maze (MTM task, a robust paradigm for evaluation of spatial learning.Trained or untrained (yoked control C57BL/6J male mice (n=10/group were subjected to the MTM task and sacrificed 6 hours following their performance. Hippocampi were taken, membrane proteins extracted and run on blue native PAGE followed by immunoblotting with specific antibodies against GABAB1, GABAB1a and GABAB2. Immunoprecipitation with subsequent mass spectrometric identification of co-precipitates was carried out to show if GABAB1 and GABAB2 as well as other interacting proteins co-precipitate. An antibody shift assay (ASA and a proximity ligation assay (PLA were also used to see if the two GABAB subunits are present in the receptor complex.Single bands were observed on Western blots, each representing GABAB1, GABAB1a or GABAB2 at an apparent molecular weight of approximately 100 kDa. Subsequently, densitometric analysis revealed that levels of GABAB1 and GABAB1a but not GABAB2- containing receptor complexes were significantly higher in trained than untrained groups. Immunoprecipitation followed by mass spectrometric studies confirmed the presence of GABAB1, GABAB2, calcium calmodulin kinases I and II, GluA1 and GluA2 as constituents of the complex. ASA and PLA also showed the presence of the two subunits of GABAB receptor within the complex. It is shown that increased levels of GABAB1 subunit-containing complexes are paralleling performance in a land maze.

  14. A selective role for α3 subunit glycine receptors in inflammatory pain

    Directory of Open Access Journals (Sweden)

    Victoria L Harvey

    2009-11-01

    Full Text Available GlyR α3 has previously been found to play a critical role in pain hypersensitivity following spinal PGE2 injection, complete Freund’s adjuvant (CFA and zymosan induced peripheral inflammation. In this study, although all models displayed typical phenotypic behaviours, no significant differences were observed when comparing the pain behaviours of Glra3-/- and wild-type littermates following the injection of capsaicin, carrageenan, kaolin/ carrageenan or monosodium iodoacetate, models of rheumatoid and osteoarthritis, respectively. However, clear differences were observed following CFA injection (p < 0.01. No significant differences were observed in the pain behaviours of Glra3-/- and wild-type littermates following experimentally induced neuropathic pain (partial sciatic nerve ligation. Similarly, Glra3-/- and wild-type littermates displayed indistinguishable visceromotor responses to colorectal distension (a model of visceral pain and in vivo spinal cord dorsal horn electrophysiology revealed no differences in responses to multimodal suprathreshold stimuli, intensities which equate to higher pain scores such as those reported in the clinic. These data suggest that apart from its clear role in CFA- and zymosan-induced pain sensitisation, hypersensitivity associated with other models of inflammation, neuropathy and visceral disturbances involves mechanisms other than the EP2 receptor - GlyR α3 pathway.

  15. The nicotinic α6 subunit gene determines variability in chronic pain sensitivity via cross-inhibition of P2X2/3 receptors

    DEFF Research Database (Denmark)

    Wieskopf, Jeffrey S; Mathur, Jayanti; Limapichat, Walrati

    2015-01-01

    expression levels of Chrna6, which encodes the α6 subunit of the nicotinic acetylcholine receptor (nAChR), as highly associated with allodynia. We confirmed the importance of α6* (α6-containing) nAChRs by analyzing both gain- and loss-of-function mutants. We find that mechanical allodynia associated...... with neuropathic and inflammatory injuries is significantly altered in α6* mutants, and that α6* but not α4* nicotinic receptors are absolutely required for peripheral and/or spinal nicotine analgesia. Furthermore, we show that Chrna6's role in analgesia is at least partially due to direct interaction and cross...

  16. A neuroligin-1-derived peptide stimulates phosphorylation of the NMDA receptor NR1 subunit and rescues MK-801-induced decrease in long-term potentiation and memory impairment

    DEFF Research Database (Denmark)

    Korshunova, Irina; Gjørlund, Michelle D; Jacobsen, Sylwia Owczarek

    2015-01-01

    neurolide-1 effects on short- and long-term social and spatial memory in social recognition, Morris water-maze, and Y-maze tests. We found that subcutaneous neurolide-1 administration, restored hippocampal LTP compromised by NMDA receptor inhibitor MK-801. It counteracted MK-801-induced memory deficit...... in the water-maze and Y-maze tests after long-term treatment (24 h and 1-2 h before the test), but not after short-term exposure (1-2 h). Long-term exposure to neurolide-1 also facilitated social recognition memory. In addition, neurolide-1-induced phosphorylation of the NMDA receptor NR1 subunit on a site...... receptor phosphorylation after treatment with NL1 or a mimetic peptide, neurolide-1, was quantified by immunoblotting. Subsequently, we investigated effects of neurolide-1 on long-term potentiation (LTP) induction in hippocampal slices compromised by NMDA receptor inhibitor MK-801. Finally, we investigated...

  17. Breaking tolerance in transgenic mice expressing the human TSH receptor A-subunit: thyroiditis, epitope spreading and adjuvant as a 'double edged sword'.

    Science.gov (United States)

    McLachlan, Sandra M; Aliesky, Holly A; Chen, Chun-Rong; Chong, Gao; Rapoport, Basil

    2012-01-01

    Transgenic mice with the human thyrotropin-receptor (TSHR) A-subunit targeted to the thyroid are tolerant of the transgene. In transgenics that express low A-subunit levels (Lo-expressors), regulatory T cell (Treg) depletion using anti-CD25 before immunization with adenovirus encoding the A-subunit (A-sub-Ad) breaks tolerance, inducing extensive thyroid lymphocytic infiltration, thyroid damage and antibody spreading to other thyroid proteins. In contrast, no thyroiditis develops in Hi-expressor transgenics or wild-type mice. Our present goal was to determine if thyroiditis could be induced in Hi-expressor transgenics using a more potent immunization protocol: Treg depletion, priming with Complete Freund's Adjuvant (CFA) + A-subunit protein and further Treg depletions before two boosts with A-sub-Ad. As controls, anti-CD25 treated Hi- and Lo-expressors and wild-type mice were primed with CFA+ mouse thyroglobulin (Tg) or CFA alone before A-sub-Ad boosting. Thyroiditis developed after CFA+A-subunit protein or Tg and A-sub-Ad boosting in Lo-expressor transgenics but Hi- expressors (and wild-type mice) were resistant to thyroiditis induction. Importantly, in Lo-expressors, thyroiditis was associated with the development of antibodies to the mouse TSHR downstream of the A-subunit. Unexpectedly, we observed that the effect of bacterial products on the immune system is a "double-edged sword". On the one hand, priming with CFA (mycobacteria emulsified in oil) plus A-subunit protein broke tolerance to the A-subunit in Hi-expressor transgenics leading to high TSHR antibody levels. On the other hand, prior treatment with CFA in the absence of A-subunit protein inhibited responses to subsequent immunization with A-sub-Ad. Consequently, adjuvant activity arising in vivo after bacterial infections combined with a protein autoantigen can break self-tolerance but in the absence of the autoantigen, adjuvant activity can inhibit the induction of immunity to autoantigens (like the

  18. Breaking Tolerance in Transgenic Mice Expressing the Human TSH Receptor A-Subunit: Thyroiditis, Epitope Spreading and Adjuvant as a ‘Double Edged Sword’

    Science.gov (United States)

    McLachlan, Sandra M.; Aliesky, Holly A.; Chen, Chun-Rong; Chong, Gao; Rapoport, Basil

    2012-01-01

    Transgenic mice with the human thyrotropin-receptor (TSHR) A-subunit targeted to the thyroid are tolerant of the transgene. In transgenics that express low A-subunit levels (Lo-expressors), regulatory T cell (Treg) depletion using anti-CD25 before immunization with adenovirus encoding the A-subunit (A-sub-Ad) breaks tolerance, inducing extensive thyroid lymphocytic infiltration, thyroid damage and antibody spreading to other thyroid proteins. In contrast, no thyroiditis develops in Hi-expressor transgenics or wild-type mice. Our present goal was to determine if thyroiditis could be induced in Hi-expressor transgenics using a more potent immunization protocol: Treg depletion, priming with Complete Freund's Adjuvant (CFA) + A-subunit protein and further Treg depletions before two boosts with A-sub-Ad. As controls, anti-CD25 treated Hi- and Lo-expressors and wild-type mice were primed with CFA+ mouse thyroglobulin (Tg) or CFA alone before A-sub-Ad boosting. Thyroiditis developed after CFA+A-subunit protein or Tg and A-sub-Ad boosting in Lo-expressor transgenics but Hi- expressors (and wild-type mice) were resistant to thyroiditis induction. Importantly, in Lo-expressors, thyroiditis was associated with the development of antibodies to the mouse TSHR downstream of the A-subunit. Unexpectedly, we observed that the effect of bacterial products on the immune system is a “double-edged sword”. On the one hand, priming with CFA (mycobacteria emulsified in oil) plus A-subunit protein broke tolerance to the A-subunit in Hi-expressor transgenics leading to high TSHR antibody levels. On the other hand, prior treatment with CFA in the absence of A-subunit protein inhibited responses to subsequent immunization with A-sub-Ad. Consequently, adjuvant activity arising in vivo after bacterial infections combined with a protein autoantigen can break self-tolerance but in the absence of the autoantigen, adjuvant activity can inhibit the induction of immunity to autoantigens (like the

  19. Deletion of the GluA1 AMPA receptor subunit impairs recency-dependent object recognition memory

    Science.gov (United States)

    Sanderson, David J.; Hindley, Emma; Smeaton, Emily; Denny, Nick; Taylor, Amy; Barkus, Chris; Sprengel, Rolf; Seeburg, Peter H.; Bannerman, David M.

    2011-01-01

    Deletion of the GluA1 AMPA receptor subunit impairs short-term spatial recognition memory. It has been suggested that short-term recognition depends upon memory caused by the recent presentation of a stimulus that is independent of contextual–retrieval processes. The aim of the present set of experiments was to test whether the role of GluA1 extends to nonspatial recognition memory. Wild-type and GluA1 knockout mice were tested on the standard object recognition task and a context-independent recognition task that required recency-dependent memory. In a first set of experiments it was found that GluA1 deletion failed to impair performance on either of the object recognition or recency-dependent tasks. However, GluA1 knockout mice displayed increased levels of exploration of the objects in both the sample and test phases compared to controls. In contrast, when the time that GluA1 knockout mice spent exploring the objects was yoked to control mice during the sample phase, it was found that GluA1 deletion now impaired performance on both the object recognition and the recency-dependent tasks. GluA1 deletion failed to impair performance on a context-dependent recognition task regardless of whether object exposure in knockout mice was yoked to controls or not. These results demonstrate that GluA1 is necessary for nonspatial as well as spatial recognition memory and plays an important role in recency-dependent memory processes. PMID:21378100

  20. Altered gamma oscillations during pregnancy through loss of δ subunit-containing GABAA receptors on parvalbumin interneurons

    Directory of Open Access Journals (Sweden)

    Isabella eFerando

    2013-09-01

    Full Text Available Gamma (γ oscillations (30-120 Hz, an emergent property of neuronal networks, correlate with memory, cognition and encoding. In the hippocampal CA3 region, locally generated γ oscillations emerge through feedback between inhibitory parvalbumin-positive basket cells (PV+BCs and the principal (pyramidal cells. PV+BCs express δ-subunit-containing GABAARs (-GABAARs and NMDA receptors (NMDA-Rs that balance the frequency of γ oscillations. Neuroactive steroids (NS, such as the progesterone-derived (3α,5α-3-hydroxy-pregnan-20-one (allopregnanolone; ALLO, modulate the expression of δ-GABAARs and the tonic conductance they mediate. Pregnancy produces large increases in ALLO and brain-region-specific homeostatic changes in δ-GABAARs expression. Here we show that in CA3, where most PV+ interneurons (INs express δ-GABAARs, expression of δ-GABAARs on INs diminishes during pregnancy, but reverts to control levels within 48 hours postpartum. These anatomical findings were corroborated by a pregnancy-related increase in the frequency of kainate-induced CA3 γ oscillations in vitro that could be countered by the NMDA-R antagonists D-AP5 and PPDA. Mimicking the typical hormonal conditions during pregnancy by supplementing 100 nM ALLO lowered the γ frequencies to levels found in virgin or postpartum mice. Our findings show that states of altered NS levels (e.g., pregnancy may provoke perturbations in γ oscillatory activity through direct effects on the GABAergic system, and underscore the importance of δ-GABAARs homeostatic plasticity in maintaining constant network output despite large hormonal changes. Inaccurate coupling of NS levels to δ-GABAAR expression may facilitate abnormal neurological and psychiatric conditions such as epilepsy, post-partum depression, and post-partum psychosis, thus providing insights into potential new treatments.

  1. Zebrafish bandoneon mutants display behavioral defects due to a mutation in the glycine receptor β-subunit

    Science.gov (United States)

    Hirata, Hiromi; Saint-Amant, Louis; Downes, Gerald B.; Cui, Wilson W.; Zhou, Weibin; Granato, Michael; Kuwada, John Y.

    2005-01-01

    Bilateral alternation of muscle contractions requires reciprocal inhibition between the two sides of the hindbrain and spinal cord, and disruption of this inhibition should lead to simultaneous activation of bilateral muscles. At 1 day after fertilization, wild-type zebrafish respond to mechanosensory stimulation with multiple fast alternating trunk contractions, whereas bandoneon (beo) mutants contract trunk muscles on both sides simultaneously. Similar simultaneous contractions are observed in wild-type embryos treated with strychnine, a blocker of the inhibitory glycine receptor (GlyR). This result suggests that glycinergic synaptic transmission is defective in beo mutants. Muscle voltage recordings confirmed that muscles on both sides of the trunk in beo are likely to receive simultaneous synaptic input from the CNS. Recordings from motor neurons revealed that glycinergic synaptic transmission was missing in beo mutants. Furthermore, immunostaining with an antibody against GlyR showed clusters in wild-type neurons but not in beo neurons. These data suggest that the failure of GlyRs to aggregate at synaptic sites causes impairment of glycinergic transmission and abnormal behavior in beo mutants. Indeed, mutations in the GlyR β-subunit, which are thought to be required for proper localization of GlyRs, were identified as the basis for the beo mutation. These data demonstrate that GlyRβ is essential for physiologically relevant clustering of GlyRs in vivo. Because GlyR mutations in humans lead to hyperekplexia, a motor disorder characterized by startle responses, the zebrafish beo mutant should be a useful animal model for this condition. PMID:15928085

  2. Ovarian cycle-linked plasticity of δ-GABAA receptor subunits in hippocampal interneurons affects γ oscillations in vivo

    Directory of Open Access Journals (Sweden)

    Albert Miklos Barth

    2014-08-01

    Full Text Available GABAA receptors containing δ subunits (δ-GABAARs are GABA-gated ion channels with extra- and perisynaptic localization, strong sensitivity to neurosteroids (NS, and a high degree of plasticity. In selective brain regions they are expressed on specific principal cells and interneurons (INs, and generate a tonic conductance that controls neuronal excitability and oscillations. Plasticity of δ-GABAARs in principal cells has been described during states of altered NS synthesis including acute stress, puberty, ovarian cycle, pregnancy and the postpartum period, with direct consequences on neuronal excitability and network dynamics. The defining network events implicated in cognitive function, memory formation and encoding are γ oscillations (30-120 Hz, a well-timed loop of excitation and inhibition between principal cells and PV-expressing INs (PV+INs. The δ-GABAARs of INs can modify γ oscillations, and a lower expression of δ-GABAARs on INs during pregnancy alters γ frequency recorded in vitro. The ovarian cycle is another physiological event with large fluctuations in NS levels and δ-GABAARs. Stages of the cycle are paralleled by swings in memory performance, cognitive function, and mood in both humans and rodents. Here we show δ-GABAARs changes during the mouse ovarian cycle in hippocampal cell types, with enhanced expression during diestrus in principal cells and specific INs. The plasticity of δ-GABAARs on PV-INs decreases the magnitude of γ oscillations continuously recorded in area CA1 throughout several days in vivo during diestrus and increases it during estrus. Such recurring changes in γ magnitude were not observed in non-cycling wild-type (WT females, cycling females lacking δ-GABAARs only on PV-INs (PV-Gabrd-/-, and in male mice during a time course equivalent to the ovarian cycle. Our findings may explain the impaired memory and cognitive performance experienced by women with premenstrual syndrome (PMS or premenstrual

  3. Propranolol decreases retention of fear memory by modulating the stability of surface glutamate receptor GluA1 subunits in the lateral amygdala.

    Science.gov (United States)

    Zhou, Jun; Luo, Yi; Zhang, Jie-Ting; Li, Ming-Xing; Wang, Can-Ming; Guan, Xin-Lei; Wu, Peng-Fei; Hu, Zhuang-Li; Jin, You; Ni, Lan; Wang, Fang; Chen, Jian-Guo

    2015-11-01

    Posttraumatic stress disorder (PTSD) is a mental disorder with enhanced retention of fear memory and has profound impact on quality of life for millions of people worldwide. The β-adrenoceptor antagonist propranolol has been used in preclinical and clinical studies for the treatment of PTSD, but the mechanisms underlying its potential efficacy on fear memory retention remain to be elucidated. We investigated the action of propranolol on the retention of conditioned fear memory, the surface expression of glutamate receptor GluA1 subunits of AMPA receptors and synaptic adaptation in the lateral amygdala (LA) of rats. Propranolol attenuated reactivation-induced strengthening of fear retention while reducing enhanced surface expression of GluA1 subunits and restoring the impaired long-term depression in LA. These effects of propranolol were mediated by antagonizing reactivation-induced enhancement of adrenergic signalling, which activates PKA and calcium/calmodulin-dependent protein kinase II and then regulates the trafficking of AMPA receptors via phosphorylation of GluA1 subunits at the C-terminus. Both i.p. injection and intra-amygdala infusion of propranolol attenuated reactivation-induced enhancement of fear retention. Reactivation strengthens fear retention by increasing the level of noradrenaline and promotes the surface expression of GluA1 subunits and the excitatory synaptic transmission in LA. These findings uncover one mechanism underlying the efficiency of propranolol on retention of fear memories and suggest that β-adrenoceptor antagonists, which act centrally, may be more suitable for the treatment of PTSD. © 2015 The British Pharmacological Society.

  4. The effect of NMDA-NR2B receptor subunit over-expression on olfactory memory task performance in the mouse.

    Science.gov (United States)

    White, Theresa L; Youngentob, Steven L

    2004-09-17

    The N-methyl-D-aspartate (NMDA) receptor in the forebrain is thought to modulate some forms of memory formation, with the NR2B subunit being particularly relevant to this process. Relative to wild-type mice, transgenic animals in which the NR2B subunit was over-expressed demonstrate superior memory in a number of behavioral tasks, including object recognition [Nature 401 (1999) 63]. The purpose of the present study was to explore the generality of such phenomena, interpreted as the effect of increasing NR2B expression on the retention of other types of sensory-related information. To accomplish this, we focused our evaluation on the highly salient sensory modality of olfaction. In the first experiment, mice performed both a novel-object-recognition task identical to that performed by Tang et al. [Nature 401 (1999) 63] and a novel-odor-recognition task analogously constructed. Although the results of the object recognition task were consistent with the previous literature, there was no evidence of an effect of NR2B over-expression on the retention of odor recognition memory in the specific task performed. As it was possible that, unlike object recognition memory, novel odor recognition is not NMDA-receptor-dependent, a second task was designed using the social transmission of food preference paradigm. In contrast to the foregoing olfactory task, there is evidence that the latter procedure is, indeed, NMDA-dependent. The results of the second study demonstrated that transgenic mice with NR2B over-expression had a clear memory advantage in this alternative odor memory paradigm. Taken together, these results suggest the NR2B subunit is an important component in some but not all forms of olfactory memory organization. Moreover, for those functions that are NMDA-receptor-dependent, these data support the growing literature demonstrating the importance of the NR2B subunit.

  5. Changes in glutamate receptor subunits within the medulla in goats after section of the carotid sinus nerves

    Science.gov (United States)

    Miller, Justin Robert; Neumueller, Suzanne; Muere, Clarissa; Olesiak, Samantha; Pan, Lawrence; Bukowy, John D.; Daghistany, Asem O.; Hodges, Matthew R.

    2014-01-01

    The mechanisms which contribute to the time-dependent recovery of resting ventilation and the ventilatory CO2 chemoreflex after carotid body denervation (CBD) are poorly understood. Herein we tested the hypothesis that there are time-dependent changes in the expression of specific AMPA, NMDA, and/or neurokinin-1 (NK1R) receptors within respiratory-related brain stem nuclei acutely or chronically after CBD in adult goats. Brain stem tissues were collected acutely (5 days) or chronically (30 days) after sham or bilateral CBD, immunostained with antibodies targeting AMPA (GluA1 or GluA2), NMDA (GluN1), or NK-1 receptors, and optical density (OD) compared. Physiological measurement confirmed categorization of each group and showed ventilatory effects consistent with bilateral CBD (Miller et al. J Appl Physiol 115: 1088–1098, 2013). Acutely after CBD, GluA1 OD was unchanged or slightly increased, but GluA2 and GluN1 OD were reduced 15–30% within the nucleus tractus solitarius (NTS) and in other medullary respiratory nuclei. Chronically after CBD, GluA1 was reduced (P < 0.05) within the caudal NTS and in other nuclei, but there was significant recovery of GluA2 and GluN1 OD. NK1 OD was not significantly different from control after CBD. We conclude that the initial decrease in GluA2 and GluN1 after CBD likely contributes to hypoventilation and the reduced CO2 chemoreflex. The partial recovery of ventilation and the CO2 chemoreflex after CBD parallel a time-dependent return of these receptors to near control levels but likely depend upon additional initiating and maintenance factors for neuroplasticity. PMID:24790015

  6. Effects of Traumatic Stress Induced in the Juvenile Period on the Expression of Gamma-Aminobutyric Acid Receptor Type A Subunits in Adult Rat Brain

    Directory of Open Access Journals (Sweden)

    Cui Yan Lu

    2017-01-01

    Full Text Available Studies have found that early traumatic experience significantly increases the risk of posttraumatic stress disorder (PTSD. Gamma-aminobutyric acid (GABA deficits were proposed to be implicated in development of PTSD, but the alterations of GABA receptor A (GABAAR subunits induced by early traumatic stress have not been fully elucidated. Furthermore, previous studies suggested that exercise could be more effective than medications in reducing severity of anxiety and depression but the mechanism is unclear. This study used inescapable foot-shock to induce PTSD in juvenile rats and examined their emotional changes using open-field test and elevated plus maze, memory changes using Morris water maze, and the expression of GABAAR subunits (γ2, α2, and α5 in subregions of the brain in the adulthood using western blotting and immunohistochemistry. We aimed to observe the role of GABAAR subunits changes induced by juvenile trauma in the pathogenesis of subsequent PTSD in adulthood. In addition, we investigated the protective effects of exercise for 6 weeks and benzodiazepine (clonazepam for 2 weeks. This study found that juvenile traumatic stress induced chronic anxiety and spatial memory loss and reduced expression of GABAAR subunits in the adult rat brains. Furthermore, exercise led to significant improvement as compared to short-term BZ treatment.

  7. Age-dependent modifications of AMPA receptor subunit expression levels and related cognitive effects in 3xTg-AD mice

    Directory of Open Access Journals (Sweden)

    Pamela eCantanelli

    2014-08-01

    Full Text Available GluA1, GluA2, GluA3, and GluA4 are the constitutive subunits of AMPA receptors (AMPARs, the major mediators of fast excitatory transmission in the mammalian central nervous system. Most AMPARs are Ca2+-impermeable because of the presence of the GluA2 subunit. GluA2 mRNA undergoes an editing process that results in a Q to R substitution, a key factor in the regulation of AMPAR Ca2+-permeability. AMPARs lacking GluA2 or containing the unedited subunit are permeable to Ca2+ and Zn2+. The phenomenon physiologically modulates synaptic plasticity while, in pathologic conditions, leads to increased vulnerability to excitotoxic neuronal death. Given the importance of these subunits, we have therefore evaluated possible associations between changes in expression levels of AMPAR subunits and development of cognitive deficits in 3xTg-AD mice, a widely investigated transgenic mouse model of Alzheimer’s disease. With qRT-PCR, we assayed hippocampal mRNA expression levels of GluA1-4 subunits occurring in young [3 months of age (m.o.a.] and old (12 m.o.a Tg-AD mice and made comparisons with levels found in age-matched wild type (WT mice. Efficiency of GluA2 RNA editing was also analyzed. All animals were cognitively tested for short- and long-term spatial memory with the Morris Water Maze (MWM navigation task. 3xTg-AD mice showed age-dependent decreases of mRNA levels for all the AMPAR subunits, with the exception of GluA2. Editing remained fully efficient with aging in 3xTg-AD and WT mice. A one-to-one correlation analysis between MWM performances and GluA1-4 mRNA expression profiles showed negative correlations between GluA2 levels and MWM performances in young 3xTg-AD mice. On the contrary, positive correlations between GluA2 mRNA and MWM performances were found in young WT mice. Our data suggest that increases of AMPARs that contain GluA1, GluA3, and GluA4 subunits may help in maintaining cognition in pre-symptomatic 3xTg-AD mice.

  8. The nicotinic acetylcholine receptors of the parasitic nematode Ascaris suum: formation of two distinct drug targets by varying the relative expression levels of two subunits.

    Directory of Open Access Journals (Sweden)

    Sally M Williamson

    2009-07-01

    Full Text Available Parasitic nematodes are of medical and veterinary importance, adversely affecting human health and animal welfare. Ascaris suum is a gastrointestinal parasite of pigs; in addition to its veterinary significance it is a good model of the human parasite Ascaris lumbricoides, estimated to infect approximately 1.4 billion people globally. Anthelmintic drugs are essential to control nematode parasites, and nicotinic acetylcholine receptors (nAChRs on nerve and muscle are the targets of cholinergic anthelmintics such as levamisole and pyrantel. Previous genetic analyses of nematode nAChRs have been confined to Caenorhabditis elegans, which is phylogenetically distinct from Ascaris spp. and many other important parasites. Here we report the cloning and expression of two nAChR subunit cDNAs from A. suum. The subunits are very similar in sequence to C. elegans UNC-29 and UNC-38, are expressed on muscle cells and can be expressed robustly in Xenopus oocytes to form acetylcholine-, nicotine-, levamisole- and pyrantel-sensitive channels. We also demonstrate that changing the stoichiometry of the receptor by injecting different ratios of the subunit cRNAs can reproduce two of the three pharmacological subtypes of nAChR present in A. suum muscle cells. When the ratio was 5:1 (Asu-unc-38ratioAsu-unc-29, nicotine was a full agonist and levamisole was a partial agonist, and oocytes responded to oxantel, but not pyrantel. At the reverse ratio (1:5 Asu-unc-38ratioAsu-unc-29, levamisole was a full agonist and nicotine was a partial agonist, and the oocytes responded to pyrantel, but not oxantel. These results represent the first in vitro expression of any parasitic nicotinic receptor and show that their properties are substantially different from those of C. elegans. The results also show that changing the expression level of a single receptor subunit dramatically altered the efficacy of some anthelmintic drugs. In vitro expression of these subunits may permit the

  9. Time-Dependent Alterations in the Expression of NMDA Receptor Subunits along the Dorsoventral Hippocampal Axis in an Animal Model of Nascent Psychosis.

    Science.gov (United States)

    Dubovyk, Valentyna; Manahan-Vaughan, Denise

    2018-04-10

    Psychosis is a mental condition that is characterized by hallucinations, delusions, disordered thought, as well as socio-emotional and cognitive impairments. Once developed, it tends to progress into a chronic psychotic illness. Here, the duration of untreated psychosis plays a crucial role: the earlier the treatment begins, relative to the first episode of the disease, the better the patient's functional prognosis. To what extent the success of early interventions relate to progressive changes at the neurotransmitter receptor level is as yet unclear. In fact, very little is known as to how molecular changes develop, transform, and become established following the first psychotic event. One neurotransmitter receptor for which a specific role in psychosis has been discussed is the N-methyl-d-aspartate receptor (NMDAR). This receptor is especially important for information encoding in the hippocampus. The hippocampus is one of the loci of functional change in psychosis, to which a role in the pathophysiology of psychosis has been ascribed. Here, we examined whether changes in NMDAR subunit expression occur along the dorsoventral axis of the hippocampus 1 week and 3 months after systemic treatment with an NMDAR antagonist (MK801) that initiates a psychosis-like state in adult rats. We found early (1 week) upregulation of the GluN2B levels in the dorso-intermediate hippocampus and late (3 month) downregulation of GluN2A expression across the entire CA1 region. The ventral hippocampus did not exhibit subunit expression changes. These data suggest that a differing vulnerability of the hippocampal longitudinal axis may occur in response to MK801-treatment and provide a time-resolved view of the putative development of pathological changes of NMDAR subunit expression in the hippocampus that initiate with an emulated first episode and progress through to the chronic stabilization of a psychosis-like state in rodents.

  10. Loss of GluN2D subunit results in social recognition deficit, social stress, 5-HT2C receptor dysfunction, and anhedonia in mice.

    Science.gov (United States)

    Yamamoto, Hideko; Kamegaya, Etsuko; Hagino, Yoko; Takamatsu, Yukio; Sawada, Wakako; Matsuzawa, Maaya; Ide, Soichiro; Yamamoto, Toshifumi; Mishina, Masayoshi; Ikeda, Kazutaka

    2017-01-01

    The N-methyl-d-aspartate (NMDA) receptor channel is involved in various physiological functions, including learning and memory. The GluN2D subunit of the NMDA receptor has low expression in the mature brain, and its role is not fully understood. In the present study, the effects of GluN2D subunit deficiency on emotional and cognitive function were investigated in GluN2D knockout (KO) mice. We found a reduction of motility (i.e., a depressive-like state) in the tail suspension test and a reduction of sucrose preference (i.e., an anhedonic state) in GluN2D KO mice that were group-housed with littermates. Despite apparently normal olfactory function and social interaction, GluN2D KO mice exhibited a decrease in preference for social novelty, suggesting a deficit in social recognition or memory. Golgi-Cox staining revealed a reduction of the complexity of dendritic trees in the accessory olfactory bulb in GluN2D KO mice, suggesting a deficit in pheromone processing pathway activation, which modulates social recognition. The deficit in social recognition may result in social stress in GluN2D KO mice. Isolation housing is a procedure that has been shown to reduce stress in mice. Interestingly, 3-week isolation and treatment with agomelatine or the 5-hydroxytryptamine-2C (5-HT 2C ) receptor antagonist SB242084 reversed the anhedonic-like state in GluN2D KO mice. In contrast, treatment with the 5-HT 2C receptor agonist CP809101 induced depressive- and anhedonic-like states in isolated GluN2D KO mice. These results suggest that social stress that is caused by a deficit in social recognition desensitizes 5-HT 2c receptors, followed by an anhedonic- and depressive-like state, in GluN2D KO mice. The GluN2D subunit of the NMDA receptor appears to be important for the recognition of individuals and development of normal emotionality in mice. 5-HT 2C receptor antagonism may be a therapeutic target for treating social stress-induced anhedonia. This article is part of the Special

  11. Cysteine protease antigens cleave CD123, the α subunit of murine IL-3 receptor, on basophils and suppress IL-3-mediated basophil expansion

    International Nuclear Information System (INIS)

    Nishikado, Hideto; Fujimura, Tsutomu; Taka, Hikari; Mineki, Reiko; Ogawa, Hideoki; Okumura, Ko; Takai, Toshiro

    2015-01-01

    Th2 type immune responses are essential for protective immunity against parasites and play crucial roles in allergic disorders. Helminth parasites secrete a variety of proteases for their infectious cycles including for host entry, tissue migration, and suppression of host immune effector cell function. Furthermore, a number of pathogen-derived antigens, as well as allergens such as papain, belong to the family of cysteine proteases. Although the link between protease activity and Th2 type immunity is well documented, the mechanisms by which proteases regulate host immune responses are largely unknown. Here, we demonstrate that the cysteine proteases papain and bromelain selectively cleave the α subunit of the IL-3 receptor (IL-3Rα/CD123) on the surface of murine basophils. The decrease in CD123 expression on the cell surface, and the degradation of the extracellular domain of recombinant CD123 were dependent on the protease activity of papain and bromelain. Pre-treatment of murine basophils with papain resulted in inhibition of IL-3-IL-3R signaling and suppressed IL-3- but not thymic stromal lymphopoietin-induced expansion of basophils in vitro. Our unexpected findings illuminate a novel mechanism for the regulation of basophil functions by protease antigens. Because IL-3 plays pivotal roles in the activation and proliferation of basophils and in protective immunity against helminth parasites, pathogen-derived proteases might contribute to the pathogenesis of infections by regulating IL-3-mediated functions in basophils. - Highlights: • We identified the murine IL3R as a novel target of papain-family cysteine proteases. • Papain-family cysteine proteases cleaved IL3Rα/CD123 on murine basophils. • Papain suppressed IL3- but not TSLP-induced expansion of murine basophils. • The inactivation of IL3R might be a strategy for pathogens to suppress host immunity

  12. Cysteine protease antigens cleave CD123, the α subunit of murine IL-3 receptor, on basophils and suppress IL-3-mediated basophil expansion

    Energy Technology Data Exchange (ETDEWEB)

    Nishikado, Hideto [Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, Tokyo (Japan); Fujimura, Tsutomu; Taka, Hikari; Mineki, Reiko [Laboratory of Proteomics and Biomolecular Science, BioMedical Research Center, Juntendo University Graduate School of Medicine, Tokyo (Japan); Ogawa, Hideoki; Okumura, Ko [Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, Tokyo (Japan); Takai, Toshiro, E-mail: t-takai@juntendo.ac.jp [Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, Tokyo (Japan)

    2015-05-01

    Th2 type immune responses are essential for protective immunity against parasites and play crucial roles in allergic disorders. Helminth parasites secrete a variety of proteases for their infectious cycles including for host entry, tissue migration, and suppression of host immune effector cell function. Furthermore, a number of pathogen-derived antigens, as well as allergens such as papain, belong to the family of cysteine proteases. Although the link between protease activity and Th2 type immunity is well documented, the mechanisms by which proteases regulate host immune responses are largely unknown. Here, we demonstrate that the cysteine proteases papain and bromelain selectively cleave the α subunit of the IL-3 receptor (IL-3Rα/CD123) on the surface of murine basophils. The decrease in CD123 expression on the cell surface, and the degradation of the extracellular domain of recombinant CD123 were dependent on the protease activity of papain and bromelain. Pre-treatment of murine basophils with papain resulted in inhibition of IL-3-IL-3R signaling and suppressed IL-3- but not thymic stromal lymphopoietin-induced expansion of basophils in vitro. Our unexpected findings illuminate a novel mechanism for the regulation of basophil functions by protease antigens. Because IL-3 plays pivotal roles in the activation and proliferation of basophils and in protective immunity against helminth parasites, pathogen-derived proteases might contribute to the pathogenesis of infections by regulating IL-3-mediated functions in basophils. - Highlights: • We identified the murine IL3R as a novel target of papain-family cysteine proteases. • Papain-family cysteine proteases cleaved IL3Rα/CD123 on murine basophils. • Papain suppressed IL3- but not TSLP-induced expansion of murine basophils. • The inactivation of IL3R might be a strategy for pathogens to suppress host immunity.

  13. Pharmacological characterisation of α6β4* nicotinic acetylcholine receptors assembled from three different α6/α3 subunit chimeras in tsA201 cells

    DEFF Research Database (Denmark)

    Jensen, Anne Bjørnskov; Hoestgaard-Jensen, Kirsten; Jensen, Anders A.

    2014-01-01

    by their inefficient functional expression in vitro. In the present study we have characterized and compared the pharmacological properties displayed by α6β4 and α6β4β3 nicotinic acetylcholine receptors assembled in tsA201 cells from the classical α6/α3 chimera (C1) and two novel α6/α3 chimeras (C6F223L and C16F223L...... should be made keeping the molecular modifications in the α6 surrogate subunits in mind, this study sheds light on the pharmacological properties of α6β4⁎ nicotinic acetylcholine receptors and demonstrates the applicability of the C6F223L and C16F223L chimeras for studies of these receptors....

  14. Identification of the subunit of cAMP receptor protein (CRP) that functionally interacts with CytR in CRP-CytR-mediated transcriptional repression

    DEFF Research Database (Denmark)

    Meibom, K L; Kallipolitis, B H; Ebright, R H

    2000-01-01

    At promoters of the Escherichia coli CytR regulon, the cAMP receptor protein (CRP) interacts with the repressor CytR to form transcriptionally inactive CRP-CytR-promoter or (CRP)(2)-CytR-promoter complexes. Here, using "oriented heterodimer" analysis, we show that only one subunit of the CRP dimer......, the subunit proximal to CytR, functionally interacts with CytR in CRP-CytR-promoter and (CRP)(2)-CytR-promoter complexes. Our results provide information about the architecture of CRP-CytR-promoter and (CRP)(2)-CytR-promoter complexes and rule out the proposal that masking of activating region 2 of CRP...

  15. Molecular determinants of desensitization and assembly of the chimeric GABA(A) receptor subunits (alpha1/gamma2) and (gamma2/alpha1) in combinations with beta2 and gamma2

    DEFF Research Database (Denmark)

    Elster, L; Kristiansen, U; Pickering, D S

    2001-01-01

    Two gamma-aminobutyric acid(A) (GABA(A)) receptor chimeras were designed in order to elucidate the structural requirements for GABA(A) receptor desensitization and assembly. The (alpha1/gamma2) and (gamma2/alpha1) chimeric subunits representing the extracellular N-terminal domain of alpha1 or gamma......, as opposed to the staining of the (gamma2/alpha1)-containing receptors, which was only slightly higher than background. To explain this, the (alpha1/gamma2) and (gamma2/alpha1) chimeras may act like alpha1 and gamma2 subunits, respectively, indicating that the extracellular N-terminal segment is important...... for assembly. However, the (alpha1/gamma2) chimeric subunit had characteristics different from the alpha1 subunit, since the (alpha1/gamma2) chimera gave rise to no desensitization after GABA stimulation in whole-cell patch-clamp recordings, which was independent of whether the chimera was expressed...

  16. Intramolecular ex vivo Fluorescence Resonance Energy Transfer (FRET of Dihydropyridine Receptor (DHPR β1a Subunit Reveals Conformational Change Induced by RYR1 in Mouse Skeletal Myotubes.

    Directory of Open Access Journals (Sweden)

    Dipankar Bhattacharya

    Full Text Available The dihydropyridine receptor (DHPR β1a subunit is essential for skeletal muscle excitation-contraction coupling, but the structural organization of β1a as part of the macromolecular DHPR-ryanodine receptor type I (RyR1 complex is still debatable. We used fluorescence resonance energy transfer (FRET to probe proximity relationships within the β1a subunit in cultured skeletal myotubes lacking or expressing RyR1. The fluorescein biarsenical reagent FlAsH was used as the FRET acceptor, which exhibits fluorescence upon binding to specific tetracysteine motifs, and enhanced cyan fluorescent protein (CFP was used as the FRET donor. Ten β1a reporter constructs were generated by inserting the CCPGCC FlAsH binding motif into five positions probing the five domains of β1a with either carboxyl or amino terminal fused CFP. FRET efficiency was largest when CCPGCC was positioned next to CFP, and significant intramolecular FRET was observed for all constructs suggesting that in situ the β1a subunit has a relatively compact conformation in which the carboxyl and amino termini are not extended. Comparison of the FRET efficiency in wild type to that in dyspedic (lacking RyR1 myotubes revealed that in only one construct (H458 CCPGCC β1a -CFP FRET efficiency was specifically altered by the presence of RyR1. The present study reveals that the C-terminal of the β1a subunit changes conformation in the presence of RyR1 consistent with an interaction between the C-terminal of β1a and RyR1 in resting myotubes.

  17. The GluN2B subunit represents a major functional determinant of NMDA receptors in human induced pluripotent stem cell-derived cortical neurons

    Directory of Open Access Journals (Sweden)

    Ioana Neagoe

    2018-04-01

    Full Text Available Abnormal signaling pathways mediated by N-methyl-d-aspartate receptors (NMDARs have been implicated in the pathogenesis of various CNS disorders and have been long considered as promising points of therapeutic intervention. However, few efforts have been previously described concerning evaluation of therapeutic modulators of NMDARs and their downstream pathways in human neurons with endogenous expression of NMDARs. In the present study, we assessed expression, functionality, and subunit composition of endogenous NMDARs in human induced pluripotent stem cell (hiPSC-derived cortical neurons (iCell Neurons and iCell GlutaNeurons. We initially confirmed the expected pharmacological response of iCell Neurons and iCell GlutaNeurons to NMDA by patch-clamp recordings. Subsequent pharmacological interrogation using GluN2 subunit-selective antagonists revealed the predominance of GluN2B in both iCell Neurons and iCell GlutaNeurons. This observation was also supported by qRT-PCR and Western blot analyses of GluN2 subunit expression as well as pharmacological experiments using positive allosteric modulators with distinct GluN2 subunit selectivity. We conclude that iCell Neurons and iCell GlutaNeurons express functional GluN2B-containing NMDARs and could serve as a valuable system for development and validation of GluN2B-modulating pharmaceutical agents. Keywords: Human induced pluripotent stem cell-derived neurons, iCell Neurons, iCell GlutaNeurons, NMDA receptors, GluN2B, Positive allosteric modulators

  18. Interleukin-2 and subunit alpha of its soluble receptor in autoimmune Addison's disease--an association study and expression analysis.

    Science.gov (United States)

    Fichna, Marta; Żurawek, Magdalena; Bratland, Eirik; Husebye, Eystein S; Kasperlik-Załuska, Anna; Czarnocka, Barbara; Januszkiewicz-Lewandowska, Danuta; Nowak, Jerzy

    2015-03-01

    Autoimmune Addison's disease (AAD) results from T cell-mediated destruction of the adrenal cortex, commonly accompanied by autoantibodies to 21-hydroxylase (21OH). In order to gain insight into the obscure aetiology of this disease, we investigated the roles of the IL2 and IL2RA genes, encoding interleukin-2 and subunit alpha of its receptor (IL2Ra), respectively. The association of AAD with IL2 and IL2RA polymorphisms (rs6822844, rs2069762, rs3136534, rs11594656, rs3118470 and rs2104286) was tested in 223 patients and 672 healthy controls. Functional studies consisted of gene expression analysis in cultured PBMCs exposed to 21OH and evaluation of serum interleukin by ELISA assays. The frequency of the minor C allele of rs3136534 was significantly decreased in AAD subjects compared to controls (OR 0.71; 95%CI 0.561-0.887; p = 0.003). Only AAD cells responded to 21OH with an elevated IL2 and IL2RA mRNA synthesis (p = 0.004 and p = 0.009 versus controls, respectively), paralleled by increased supernatant levels of both cytokines (p = 0.031 and p = 0.001 versus controls). IL2 mRNA level in 21OH-stimulated AAD PBMCs correlated negatively with age (p = 0.036) and positively with serum antibodies to 21OH (p = 0.006). Carriers of the rs2104286 AA genotype demonstrated higher IL2RA mRNA (p = 0.022) and soluble IL2Ra secretion (p = 0.029) upon 21OH stimulation. Serum interleukin-2 in AAD subjects was significantly higher compared to controls (4.61 ± 4.3 versus 1.71 ± 3.2 pg/mL, p < 0.001), whereas sIL2Ra levels remained similar in both groups (p = 0.885). In conclusion, the study reveals an association between AAD and IL2 locus. It confirms specific 21OH-directed reactivity of the peripheral AAD lymphocytes, which display increased synthesis of interleukin-2 and sIL2Ra.

  19. Dynamic interactions of the asialoglycoprotein receptor subunits with coated pits. Enhanced interactions of H2 following association with H1.

    Science.gov (United States)

    Katzir, Z; Nardi, N; Geffen, I; Fuhrer, C; Henis, Y I

    1994-08-26

    Lateral mobility studies comparing native and mutated membrane proteins, combined with treatments that alter clathrin lattice structure, can measure membrane protein-coated pit interactions in intact cells (Fire, E., Zwart, D., Roth, M. G., and Henis, Y. I. (1991) J. Cell Biol. 115, 1585-1594). We applied this approach to study the interactions of the H1 and H2 human asialoglycoprotein receptor subunits with coated pits. The lateral mobilities of singly expressed and coexpressed H1 and H2B (the H2 species that reaches the cell surface) were measured by fluorescence photobleaching recovery. They were compared with mutant proteins, H1(5A) (Tyr-5 replaced by Ala) and H2(5A) (Phe-5 replaced by Ala). While the mobile fractions of H1, H2B, and their mutants were similar, the lateral diffusion rate (measured by D, the lateral diffusion coefficient) was significantly slower for H1, whether expressed alone or with H2B. Coexpression with H1 reduced D of H2B to that of H1. Disruption of the clathrin lattices by hypertonic medium elevated D of H1, H1(5A), H2B, and H2(5A) to the same final level, without affecting their mobile fractions. Cytosol acidification, which retains altered clathrin lattices attached to the membrane and prevents coated vesicle formation, immobilized part of the H1 molecules, reflecting stable entrapment in "frozen" coated pits. H1(5A), H2B, and H2(5A) were not affected; however, coexpression of H2B with H1 conferred the sensitivity to cytosol acidification on H2B. Our results suggest that H1 lateral mobility is inhibited by dynamic interactions with coated pits in which Tyr-5 is involved. H2B resembles H1(5A) rather than H1, and its interactions with coated pits are weaker; efficient interaction of H2B with coated pits depends on complex formation with H1.

  20. GABA receptor subunit distribution and FMRP-mGluR5 signaling abnormalities in the cerebellum of subjects with schizophrenia, mood disorders, and autism.

    Science.gov (United States)

    Fatemi, S Hossein; Folsom, Timothy D

    2015-09-01

    Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the brain. GABAergic receptor abnormalities have been documented in several major psychiatric disorders including schizophrenia, mood disorders, and autism. Abnormal expression of mRNA and protein for multiple GABA receptors has also been observed in multiple brain regions leading to alterations in the balance between excitatory/inhibitory signaling in the brain with potential profound consequences for normal cognition and maintenance of mood and perception. Altered expression of GABAA receptor subunits has been documented in fragile X mental retardation 1 (FMR1) knockout mice, suggesting that loss of its protein product, fragile X mental retardation protein (FMRP), impacts GABAA subunit expression. Recent postmortem studies from our laboratory have shown reduced expression of FMRP in the brains of subjects with schizophrenia, bipolar disorder, major depression, and autism. FMRP acts as a translational repressor and, under normal conditions, inhibits metabotropic glutamate receptor 5 (mGluR5)-mediated signaling. In fragile X syndrome (FXS), the absence of FMRP is hypothesized to lead to unregulated mGluR5 signaling, ultimately resulting in the behavioral and intellectual impairments associated with this disorder. Our laboratory has identified changes in mGluR5 expression in autism, schizophrenia, and mood disorders. In the current review article, we discuss our postmortem data on GABA receptors, FMRP, and mGluR5 levels and compare our results with other laboratories. Finally, we discuss the interactions between these molecules and the potential for new therapeutic interventions that target these interconnected signaling systems. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. T−B+NK+ severe combined immunodeficiency caused by complete deficiency of the CD3ζ subunit of the T-cell antigen receptor complex

    OpenAIRE

    Roberts, Joseph L.; Lauritsen, Jens Peter H.; Cooney, Myriah; Parrott, Roberta E.; Sajaroff, Elisa O.; Win, Chan M.; Keller, Michael D.; Carpenter, Jeffery H.; Carabana, Juan; Krangel, Michael S.; Sarzotti, Marcella; Zhong, Xiao-Ping; Wiest, David L.; Buckley, Rebecca H.

    2007-01-01

    CD3ζ is a subunit of the T-cell antigen receptor (TCR) complex required for its assembly and surface expression that also plays an important role in TCR-mediated signal transduction. We report here a patient with T−B+NK+ severe combined immunodeficiency (SCID) who was homozygous for a single C insertion following nucleotide 411 in exon 7 of the CD3ζ gene. The few T cells present contained no detectable CD3ζ protein, expressed low levels of cell surface CD3ε, and were nonfunctional. CD4+CD8−CD...

  2. Functional isotypes are not encoded by the constant region genes of the beta subunit of the T cell receptor for antigen/major histocompatibility complex

    OpenAIRE

    1984-01-01

    Human T cell clones and a cDNA probe specific for constant regions of the beta subunit of the antigen/major histocompatibility complex (MHC) receptor, TiC beta 1 and TiC beta 2, were employed to determine whether these genes were differentially used by functional classes of T lymphocytes. DNA from 10 interleukin-2-dependent T cell clones including class I and class II MHC-specific cytotoxic T lymphocytes (n = 6), T4+ inducer T lymphocytes (n = 2), and T8+ suppressor T lymphocytes (n = 2) show...

  3. Circadian and developmental regulation of N-methyl-d-aspartate-receptor 1 mRNA splice variants and N-methyl-d-aspartate-receptor 3 subunit expression within the rat suprachiasmatic nucleus

    DEFF Research Database (Denmark)

    Bendová, Z; Sumová, A; Mikkelsen, Jens D.

    2009-01-01

    The circadian rhythms of mammals are generated by the circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Its intrinsic period is entrained to a 24 h cycle by external cues, mainly by light. Light impinging on the SCN at night causes either advancing or delaying phase...... shifts of the circadian clock. N-methyl-d-aspartate receptors (NMDAR) are the main glutamate receptors mediating the effect of light on the molecular clockwork in the SCN. They are composed of multiple subunits, each with specific characteristics whose mutual interactions strongly determine properties...

  4. The α1, α2, α3, and γ2 subunits of GABAA receptors show characteristic spatial and temporal expression patterns in rhombencephalic structures during normal human brain development

    NARCIS (Netherlands)

    Stojanovic, Tamara; Capo, Ivan; Aronica, Eleonora; Adle-Biassette, Homa; Höger, Harald; Sieghart, Werner; Kovacs, Gabor G.; Milenkovic, Ivan

    2016-01-01

    γ-Aminobutyric acid (GABA) is the most abundant inhibitory neurotransmitter in adult mammalian brain, mediating its actions chiefly via a pentameric chloride ion channel, the GABAA receptor. Nineteen different subunits (α1-6, β1-3, γ1-3, δ, ε, π, θ, ρ1-3) can give rise to multiple receptor subtypes

  5. Identification of Interleukin-27 (IL-27)/IL-27 Receptor Subunit Alpha as a Critical Immune Axis for In Vivo HIV Control.

    Science.gov (United States)

    Ruiz-Riol, M; Berdnik, D; Llano, A; Mothe, B; Gálvez, C; Pérez-Álvarez, S; Oriol-Tordera, B; Olvera, A; Silva-Arrieta, S; Meulbroek, M; Pujol, F; Coll, J; Martinez-Picado, J; Ganoza, C; Sanchez, J; Gómez, G; Wyss-Coray, T; Brander, C

    2017-08-15

    Intact and broad immune cell effector functions and specific individual cytokines have been linked to HIV disease outcome, but their relative contribution to HIV control remains unclear. We asked whether the proteome of secreted cytokines and signaling factors in peripheral blood can be used to discover specific pathways critical for host viral control. A custom glass-based microarray, able to measure >600 plasma proteins involved in cell-to-cell communication, was used to measure plasma protein profiles in 96 HIV-infected, treatment-naive individuals with high (>50,000) or low (HIV RNA copies/ml) viral loads. Univariate and regression model analysis demonstrate that plasma levels of soluble interleukin-27 (IL-27) are significantly elevated in individuals with high plasma viremia ( P HIV-DNA copy numbers in peripheral blood mononuclear cells (PBMC) (Rho = 0.4011; P = 0.0027). Moreover, soluble IL-27 plasma levels are negatively associated with the breadth and magnitude of the total virus-specific T-cell responses and directly with plasma levels of molecules involved in Wnt/β-catenin signaling. In addition to IL-27, gene expression levels of the specific IL-27 receptor ( IL27RA ) in PBMC correlated directly with both plasma viral load (Rho = 0.3531; P = 0.0218) and the proviral copy number in the peripheral blood as an indirect measure of partial viral reservoir (Rho = 0.4580; P = 0.0030). These results were validated in unrelated cohorts of early infected subjects as well as subjects before and after initiation of antiretroviral treatment, and they identify IL-27 and its specific receptor as a critical immune axis for the antiviral immune response and as robust correlates of viral load and proviral reservoir size in PBMC. IMPORTANCE The detailed knowledge of immune mechanisms that contribute to HIV control is a prerequisite for the design of effective treatment strategies to achieve HIV cure. Cells communicate with each other by secreting signaling proteins, and

  6. Subunits Rip1p and Cox9p of the respiratory chain contribute to diclofenac-induced mitochondrial dysfunction.

    Science.gov (United States)

    van Leeuwen, Jolanda S; Orij, Rick; Luttik, Marijke A H; Smits, Gertien J; Vermeulen, Nico P E; Vos, J Chris

    2011-03-01

    The widely used drug diclofenac can cause serious heart, liver and kidney injury, which may be related to its ability to cause mitochondrial dysfunction. Using Saccharomyces cerevisiae as a model system, we studied the mechanisms of diclofenac toxicity and the role of mitochondria therein. We found that diclofenac reduced cell growth and viability and increased levels of reactive oxygen species (ROS). Strains increasingly relying on respiration for their energy production showed enhanced sensitivity to diclofenac. Furthermore, oxygen consumption was inhibited by diclofenac, suggesting that the drug inhibits respiration. To identify the site of respiratory inhibition, we investigated the effects of deletion of respiratory chain subunits on diclofenac toxicity. Whereas deletion of most subunits had no effect, loss of either Rip1p of complex III or Cox9p of complex IV resulted in enhanced resistance to diclofenac. In these deletion strains, diclofenac did not increase ROS formation as severely as in the wild-type. Our data are consistent with a mechanism of toxicity in which diclofenac inhibits respiration by interfering with Rip1p and Cox9p in the respiratory chain, resulting in ROS production that causes cell death.

  7. Molecular analysis of the interaction between the intracellular loops of the human serotonin receptor type 6 (5-HT6) and the α subunit of GS protein

    International Nuclear Information System (INIS)

    Kang, Hatan; Lee, Won Kyu; Choi, Yun Hui; Vukoti, Krishna Moorthy; Bang, Won Gi; Yu, Yeon Gyu

    2005-01-01

    The serotonin type 6 (5-HT 6 ) receptor is a G-protein coupled receptor (GPCR) coupled to a stimulatory G-protein (G S ). To identify the structural basis for the interaction of the 5-HT 6 receptor with the G S protein, we have dissected the interaction between GST-fusion proteins containing the second intracellular loop (iL2), the third intracellular loop (iL3), or the C-terminal tail of the 5-HT 6 receptor and the α subunit of G S (Gα S ). The direct interaction of iL3 and Gα S was demonstrated by co-immunoprecipitation. Furthermore, the kinetic parameters of the interaction between iL3 and Gα S were measured by surface plasmon resonance, and the apparent dissociation constant was determined to be 0.9 x 10 -6 M. In contrast, the second intracellular loop and C-terminal tail regions showed negligible affinity to Gα S . The critical residues within the iL3 region for the interaction with Gα S were identified as conserved positively charged residues near the C-terminus of iL3 by measuring the cellular levels of cAMP produced in response to 5-HT stimulation of cells transfected with 5-HT 6 receptor mutants

  8. Heterotrimeric G protein beta1gamma2 subunits change orientation upon complex formation with G protein-coupled receptor kinase 2 (GRK2) on a model membrane.

    Science.gov (United States)

    Boughton, Andrew P; Yang, Pei; Tesmer, Valerie M; Ding, Bei; Tesmer, John J G; Chen, Zhan

    2011-09-13

    Few experimental techniques can assess the orientation of peripheral membrane proteins in their native environment. Sum Frequency Generation (SFG) vibrational spectroscopy was applied to study the formation of the complex between G protein-coupled receptor (GPCR) kinase 2 (GRK2) and heterotrimeric G protein β(1)γ(2) subunits (Gβγ) at a lipid bilayer, without any exogenous labels. The most likely membrane orientation of the GRK2-Gβγ complex differs from that predicted from the known protein crystal structure, and positions the predicted receptor docking site of GRK2 such that it would more optimally interact with GPCRs. Gβγ also appears to change its orientation after binding to GRK2. The developed methodology is widely applicable for the study of other membrane proteins in situ.

  9. Involvement of N-methyl-D-aspartate receptor subunits in zinc-mediated modification of CA1 long-term potentiation in the developing hippocampus.

    Science.gov (United States)

    Takeda, Atsushi; Itagaki, Kosuke; Ando, Masaki; Oku, Naoto

    2012-03-01

    Zinc is an endogenous N-methyl-D-aspartate (NMDA) receptor blocker. It is possible that zinc-mediated modification of hippocampal CA1 long-term potentiation (LTP) is linked to the expression of NMDA receptor subunits, which varies with postnatal development. In the present study, the effect of ZnCl(2) and CaEDTA, a membrane-impermeable zinc chelator, on CA1 LTP induction was examined in hippocampal slices from immature (3-week-old) and young (6-week-old) rats. Tetanus (10-100 Hz, 1 sec)-induced CA1 LTP was more greatly enhanced in 3-week-old rats. CA1 LTP was inhibited in the presence of 2-amino-5-phosphonovalerate (APV), an NMDA receptor antagonist, and CaEDTA in 3-week-old rats, as in the case of 6-week-old rats reported previously. In 3-week-old rats, on the other hand, 5 μM ZnCl(2) attenuated NMDA receptor-mediated EPSPs more than in 6-week-old rats and significantly attenuated CA1 LTP. Moreover, 5 μM ZnCl(2) significantly attenuated CA1 LTP in the presence of (2R,4S)-4-(3-phosphonopropyl)-2-piperidinecarboxylic acid (PPPA), an NR2A antagonist, in 3-week-old rats, but not that in the presence of ifenprodil, an NR2B antagonist, suggesting that zinc-mediated attenuation of CA1 LTP is associated with the preferential expression of NR2B subunit in 3-week-old rats. In 6-week-old rats, however, 5 μM ZnCl(2) significantly potentiated CA1 LTP and also CA1 LTP in the presence of PPPA. The present study demonstrates that endogenous zinc may participate in the induction of CA1 LTP. It is likely that the changes in expression of NMDA receptor subunits are involved in the zinc-mediated modification of CA1 LTP in the developing hippocampus. Copyright © 2011 Wiley Periodicals, Inc.

  10. Difference in Perseverative Errors during a Visual Attention Task with Auditory Distractors in Alpha-9 Nicotinic Receptor Subunit Wild Type and Knock-Out Mice.

    Science.gov (United States)

    Jorratt, Pascal; Delano, Paul H; Delgado, Carolina; Dagnino-Subiabre, Alexies; Terreros, Gonzalo

    2017-01-01

    The auditory efferent system is a neural network that originates in the auditory cortex and projects to the cochlear receptor through olivocochlear (OC) neurons. Medial OC neurons make cholinergic synapses with outer hair cells (OHCs) through nicotinic receptors constituted by α9 and α10 subunits. One of the physiological functions of the α9 nicotinic receptor subunit (α9-nAChR) is the suppression of auditory distractors during selective attention to visual stimuli. In a recent study we demonstrated that the behavioral performance of alpha-9 nicotinic receptor knock-out (KO) mice is altered during selective attention to visual stimuli with auditory distractors since they made less correct responses and more omissions than wild type (WT) mice. As the inhibition of the behavioral responses to irrelevant stimuli is an important mechanism of the selective attention processes, behavioral errors are relevant measures that can reflect altered inhibitory control. Errors produced during a cued attention task can be classified as premature, target and perseverative errors. Perseverative responses can be considered as an inability to inhibit the repetition of an action already planned, while premature responses can be considered as an index of the ability to wait or retain an action. Here, we studied premature, target and perseverative errors during a visual attention task with auditory distractors in WT and KO mice. We found that α9-KO mice make fewer perseverative errors with longer latencies than WT mice in the presence of auditory distractors. In addition, although we found no significant difference in the number of target error between genotypes, KO mice made more short-latency target errors than WT mice during the presentation of auditory distractors. The fewer perseverative error made by α9-KO mice could be explained by a reduced motivation for reward and an increased impulsivity during decision making with auditory distraction in KO mice.

  11. Difference in Perseverative Errors during a Visual Attention Task with Auditory Distractors in Alpha-9 Nicotinic Receptor Subunit Wild Type and Knock-Out Mice

    Directory of Open Access Journals (Sweden)

    Pascal Jorratt

    2017-11-01

    Full Text Available The auditory efferent system is a neural network that originates in the auditory cortex and projects to the cochlear receptor through olivocochlear (OC neurons. Medial OC neurons make cholinergic synapses with outer hair cells (OHCs through nicotinic receptors constituted by α9 and α10 subunits. One of the physiological functions of the α9 nicotinic receptor subunit (α9-nAChR is the suppression of auditory distractors during selective attention to visual stimuli. In a recent study we demonstrated that the behavioral performance of alpha-9 nicotinic receptor knock-out (KO mice is altered during selective attention to visual stimuli with auditory distractors since they made less correct responses and more omissions than wild type (WT mice. As the inhibition of the behavioral responses to irrelevant stimuli is an important mechanism of the selective attention processes, behavioral errors are relevant measures that can reflect altered inhibitory control. Errors produced during a cued attention task can be classified as premature, target and perseverative errors. Perseverative responses can be considered as an inability to inhibit the repetition of an action already planned, while premature responses can be considered as an index of the ability to wait or retain an action. Here, we studied premature, target and perseverative errors during a visual attention task with auditory distractors in WT and KO mice. We found that α9-KO mice make fewer perseverative errors with longer latencies than WT mice in the presence of auditory distractors. In addition, although we found no significant difference in the number of target error between genotypes, KO mice made more short-latency target errors than WT mice during the presentation of auditory distractors. The fewer perseverative error made by α9-KO mice could be explained by a reduced motivation for reward and an increased impulsivity during decision making with auditory distraction in KO mice.

  12. Ghrelin upregulates the phosphorylation of the GluN2B subunit of the NMDA receptor by activating GHSR1a and Fyn in the rat hippocampus.

    Science.gov (United States)

    Berrout, Liza; Isokawa, Masako

    2018-01-01

    Ghrelin and its receptor GHSR1a have been shown to exert numerous physiological functions in the brain, in addition to the well-established orexigenic role in the hypothalamus. Earlier work indicated that ghrelin stimulated the phosphorylation of the GluN1 subunit of the NMDA receptor (NMDAR) and enhanced synaptic transmission in the hippocampus. In the present study, we report that the exogenous application of ghrelin increased GluN2B phosphorylation. This increase was independent of GluN2B subunit activity or NMDAR channel activity. However, it depended on the activation of GHSR1a and Fyn as it was blocked by D-Lys3-GHRP-6 and PP2, respectively. Inhibitors for G-protein-regulated second messengers, such as Rp-cAMP, H89, TBB, ryanodine, and thapsigargin, unexpectedly enhanced GluN2B phosphorylation, suggesting that cAMP, PKA, casein kinase II, and cytosolic calcium signaling may oppose to the effect of ghrelin on the phosphorylation of GluN2B. Our findings suggest that 1) GluN2B is likely a molecular target of ghrelin and GHSR1a-driven signaling cascades, and 2) the ghrelin-mediated phosphorylation of GluN2B depends on Fyn activation under complex negative regulation by other second messengers. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Distinct Contributions of Autophagy Receptors in Measles Virus Replication.

    Science.gov (United States)

    Petkova, Denitsa S; Verlhac, Pauline; Rozières, Aurore; Baguet, Joël; Claviere, Mathieu; Kretz-Remy, Carole; Mahieux, Renaud; Viret, Christophe; Faure, Mathias

    2017-05-22

    Autophagy is a potent cell autonomous defense mechanism that engages the lysosomal pathway to fight intracellular pathogens. Several autophagy receptors can recognize invading pathogens in order to target them towards autophagy for their degradation after the fusion of pathogen-containing autophagosomes with lysosomes. However, numerous intracellular pathogens can avoid or exploit autophagy, among which is measles virus (MeV). This virus induces a complete autophagy flux, which is required to improve viral replication. We therefore asked how measles virus interferes with autophagy receptors during the course of infection. We report that in addition to NDP52/CALCOCO₂ and OPTINEURIN/OPTN, another autophagy receptor, namely T6BP/TAXIBP1, also regulates the maturation of autophagosomes by promoting their fusion with lysosomes, independently of any infection. Surprisingly, only two of these receptors, NDP52 and T6BP, impacted measles virus replication, although independently, and possibly through physical interaction with MeV proteins. Thus, our results suggest that a restricted set of autophagosomes is selectively exploited by measles virus to replicate in the course of infection.

  14. Molecular cloning of a second subunit of the receptor for human granulocyte - macrophage colony-stimulating factor (GM-CSF): Reconstitution of a high-affinity GM-CSF receptor

    International Nuclear Information System (INIS)

    Hayashida, Kazuhiro; Kitamura, Toshio; Gorman, D.M.; Miyajima, Atsushi; Arai, Kenichi; Yokota, Takashi

    1990-01-01

    Using the mouse interleukin 3 (IL-3) receptor cDNA as a probe, the authors obtained a monologous cDNA (KH97) from a cDNA library of a human hemopoietic cell line, TF-1. The protein encoded by the KH97 cDNA has 56% amino acid sequence identity with the mouse IL-3 receptor and retains features common to the family of cytokine receptors. Fibroblasts transfected with the KH97 cDNA expressed a protein of 120 kDa but did not bind any human cytokines, including IL-3 and granulocyte - macrophage colony-stimulating factor (GM-CSF). Interestingly, cotransfection of cDNAs for KH97 and the low-affinity human GM-CSF receptor in fibroblasts resulted in formation of a high-affinity receptor for GM-CSF. The dissociation rate of GM-CSF from the reconstituted high-affinity receptor was slower than that from the low-affinity site, whereas the association rate was unchanged. Cross-linking of 125 I-labeled GM-CSF to fibroblasts cotransfected with both cDNAs revealed the same cross-linking patterns as in TF-1 cells - i.e., two major proteins of 80 and 120 kDa which correspond to the low-affinity GM-CSF receptor and the KH97 protein, respectively. These results indicate that the high-affinity GM-CSF receptor is composed of at least two components in a manner analogous to the IL-2 receptor. They therefore propose to designate the low-affinity GM-CSF receptor and the KH97 protein as the α and β subunits of the GM-CSF receptor, respectively

  15. Early continuous white noise exposure alters auditory spatial sensitivity and expression of GAD65 and GABAA receptor subunits in rat auditory cortex.

    Science.gov (United States)

    Xu, Jinghong; Yu, Liping; Cai, Rui; Zhang, Jiping; Sun, Xinde

    2010-04-01

    Sensory experiences have important roles in the functional development of the mammalian auditory cortex. Here, we show how early continuous noise rearing influences spatial sensitivity in the rat primary auditory cortex (A1) and its underlying mechanisms. By rearing infant rat pups under conditions of continuous, moderate level white noise, we found that noise rearing markedly attenuated the spatial sensitivity of A1 neurons. Compared with rats reared under normal conditions, spike counts of A1 neurons were more poorly modulated by changes in stimulus location, and their preferred locations were distributed over a larger area. We further show that early continuous noise rearing induced significant decreases in glutamic acid decarboxylase 65 and gamma-aminobutyric acid (GABA)(A) receptor alpha1 subunit expression, and an increase in GABA(A) receptor alpha3 expression, which indicates a returned to the juvenile form of GABA(A) receptor, with no effect on the expression of N-methyl-D-aspartate receptors. These observations indicate that noise rearing has powerful adverse effects on the maturation of cortical GABAergic inhibition, which might be responsible for the reduced spatial sensitivity.

  16. Expression of the GABA(A) receptor alpha6 subunit in cultured cerebellar granule cells is developmentally regulated by activation of GABA(A) receptors

    DEFF Research Database (Denmark)

    Carlson, B X; Belhage, B; Hansen, Gert Helge

    1997-01-01

    Da (alpha6 subunit) radioactive peaks in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In contrast, THIP-treated granule cells at 8 DIV demonstrated a small but significant decrease from control cultures in the photoincorporation of [3H]Ro15-4513 in the 51-kDa peak; however...... that the major effect of THIP was to increase alpha6 subunit clustering on granule cell bodies as well as neurites, 15-fold and sixfold, respectively. Using in situ hybridization, a small THIP-induced increase in alpha6 mRNA was detected at 4 DIV; however, no effect was apparent at 8 DIV. These data suggest...

  17. Mapping of the human NMDA receptor subunit (NMDAR1) and the proposed NMDA receptor glutamate-binding subunit (NMDARA1) to chromosomes 9q34.3 and chromosome 8, respectively

    DEFF Research Database (Denmark)

    Collins, C; Duff, C; Duncan, A M

    1993-01-01

    to human chromosome 8 using a somatic cell hybrid panel. Because the gene causing HD has been localized to chromosome 4p16.3, the chromosome assignments reported here are inconsistent with either of these genes playing a causative role in the molecular pathology of HD. However, it is noteworthy......A role for the N-methyl-D-aspartate (NMDA) receptor in the molecular pathology underlying Huntington disease (HD) has been proposed on the basis of neurochemical studies in HD and the ability of the NMDA receptor to mediate neuronal cell death. The molecular cloning of the human NMDA receptor...

  18. GABA-A receptor beta3 and alpha5 subunit gene cluster on chromosome 15q11-q13 and bipolar disorder: a genetic association study.

    Science.gov (United States)

    Papadimitriou, G N; Dikeos, D G; Karadima, G; Avramopoulos, D; Daskalopoulou, E G; Stefanis, C N

    2001-05-08

    There is accumulated evidence that the genes coding for the receptor of gamma aminobutyric acid (GABA), the most important inhibitory neurotransmitter in the CNS, may be involved in the pathogenesis of affective disorders. In a previous study, we have found a genetic association between the GABA-A receptor alpha5 subunit gene locus (GABRA5) on chromosome 15q11-of 13 and bipolar affective disorder. The aim of the present study was to examine the same subjects to see if there exists a genetic association between bipolar affective disorder and the GABA receptor beta3 subunit gene (GABRB3), which is located within 100 kb from GABRA5. The sample consisted of 48 bipolar patients compared to 44 controls (blood donors). All subjects were Greek, unrelated, and personally interviewed. Diagnosis was based on DSM-IV and ICD-10 criteria. The marker used was a dinucleotide (CA) repeat polymorphism with 12 alleles 179 to 201 bp long; genotyping was successful in all patients and 43 controls. The distribution of GABRB3 genotypes among the controls did not deviate significantly from the Hardy-Weinberg equilibrium. No differences in allelic frequencies between bipolar patients and controls were found for GABRB3, while this locus and GABRA5 did not seem to be in significant linkage disequilibrium. In conclusion, the GABRB3 CA-repeat polymorphism we investigated does not present the observed association between bipolar affective illness and GABRA5. This could be due to higher mutation rate in the GABRB3 CA-repeat polymorphism, but it might also signify that GABRA5 is the gene actually associated with the disease. Copyright 2001 Wiley-Liss, Inc.

  19. Tailoring subunit vaccine immunity with adjuvant combinations and delivery routes using the Middle East respiratory coronavirus (MERS-CoV receptor-binding domain as an antigen.

    Directory of Open Access Journals (Sweden)

    Jiaming Lan

    Full Text Available The development of an effective vaccine is critical for prevention of a Middle East respiratory syndrome coronavirus (MERS-CoV pandemic. Some studies have indicated the receptor-binding domain (RBD protein of MERS-CoV spike (S is a good candidate antigen for a MERS-CoV subunit vaccine. However, highly purified proteins are typically not inherently immunogenic. We hypothesised that humoral and cell-mediated immunity would be improved with a modification of the vaccination regimen. Therefore, the immunogenicity of a novel MERS-CoV RBD-based subunit vaccine was tested in mice using different adjuvant formulations and delivery routes. Different vaccination regimens were compared in BALB/c mice immunized 3 times intramuscularly (i.m. with a vaccine containing 10 µg of recombinant MERS-CoV RBD in combination with either aluminium hydroxide (alum alone, alum and polyriboinosinic acid (poly I:C or alum and cysteine-phosphate-guanine (CpG oligodeoxynucleotides (ODN. The immune responses of mice vaccinated with RBD, incomplete Freund's adjuvant (IFA and CpG ODN by a subcutaneous (s.c. route were also investigated. We evaluated the induction of RBD-specific humoral immunity (total IgG and neutralizing antibodies and cellular immunity (ELISpot assay for IFN-γ spot-forming cells and splenocyte cytokine production. Our findings indicated that the combination of alum and CpG ODN optimized the development of RBD-specific humoral and cellular immunity following subunit vaccination. Interestingly, robust RBD-specific antibody and T-cell responses were induced in mice immunized with the rRBD protein in combination with IFA and CpG ODN, but low level of neutralizing antibodies were elicited. Our data suggest that murine immunity following subunit vaccination can be tailored using adjuvant combinations and delivery routes. The vaccination regimen used in this study is promising and could improve the protection offered by the MERS-CoV subunit vaccine by eliciting

  20. G protein-coupled receptor kinase 2 negatively regulates chemokine signaling at a level downstream from G protein subunits

    NARCIS (Netherlands)

    Jimenez-Sainz, MC; Murga, C; Kavelaars, A; Jurado-Pueyo, M; Krakstad, BF; Heijnen, CJ; Mayor, F; Aragay, AM

    The G protein-coupled receptor kinase 2 (GRK2) phosphorylates and desensitizes ligand-activated G protein-coupled-receptors. Here, evidence is shown for a novel role of GRK2 in regulating chemokine-mediated signals. The presence of increased levels of GRK2 in human embryonic kidney (HEK) 293 cells

  1. Characterization, cell-surface expression and ligand-binding properties of different truncated N-terminal extracellular domains of the ionotropic glutamate receptor subunit GluR1.

    Science.gov (United States)

    McIlhinney, R A; Molnár, E

    1996-04-01

    To identify the location of the first transmembrane segment of the GluR1 glutamate receptor subunit artificial stop codons have been introduced into the N-terminal domain at amino acid positions 442, 510, and 563, namely just before and spanning the proposed first two transmembrane regions. The resultant truncated N-terminal fragments of GluR1, termed NT1, NT2, and NT3 respectively were expressed in Cos-7 cells and their cellular distribution and cell-surface expression analysed using an N-terminal antibody to GluR1. All of the fragments were fully glycosylated and were found to be associated with cell membranes but none was secreted. Differential extraction of the cell membranes indicated that both NT1 and NT2 behave as peripheral membrane proteins. In contrast NT3, like the full subunit, has integral membrane protein properties. Furthermore only NT3 is expressed at the cell surface as determined by immunofluorescence and cell-surface biotinylation. Protease protection assays indicated that only NT3 had a cytoplasmic tail. Binding studies using the selective ligand [(3)H]alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate ([(3)H]AMPA) demonstrated that NT3 does not bind ligand. Together these results indicate that the first transmembrane domain of the GluR1 subunit lies between residues 509 and 562, that the N-terminal domain alone cannot form a functional ligand-binding site and that this domain can be targeted to the cell surface provided that it has a transmembrane-spanning region.

  2. E3 Ubiquitin Ligase RNF125 Activates Interleukin-36 Receptor Signaling and Contributes to Its Turnover.

    Science.gov (United States)

    Saha, Siddhartha S; Caviness, Gary; Yi, Guanghui; Raymond, Ernest L; Mbow, M Lamine; Kao, C Cheng

    2018-01-01

    Signaling by the interleukin-36 receptor (IL-36R) is linked to inflammatory diseases such as psoriasis. However, the regulation of IL-36R signaling is poorly understood. Activation of IL-36R signaling in cultured cells results in an increased polyubiquitination of the receptor subunit, IL-1Rrp2. Treatment with deubiquitinases shows that the receptor subunit of IL-36R, IL-1Rrp2, is primarily polyubiquitinated at the K63 position, which is associated with endocytic trafficking and signal transduction. A minor amount of ubiquitination is at the K48 position that is associated with protein degradation. A focused siRNA screen identified RNF125, an E3 ubiquitin ligase, to ubiquitinate IL-1Rrp2 upon activation of IL-36R signaling while not affecting the activated IL-1 receptor. Knockdown of RNF125 decreases signal transduction by the IL-36R. Overexpression of RNF125 in HEK293T cells activates IL-36R signaling and increases the ubiquitination of IL-1Rrp2 and its subsequent turnover. RNF125 can coimmunoprecipitate with the IL-36R, and it traffics with IL-1Rrp2 from the cell surface to lysosomes. Mutations of Lys568 and Lys569 in the C-terminal tail of IL-1Rrp2 decrease ubiquitination by RNF125 and increase the steady-state levels of IL-1Rrp2. These results demonstrate that RNF125 has multiple regulatory roles in the signaling, trafficking, and turnover of the IL-36R. © 2017 S. Karger AG, Basel.

  3. Caloric restriction increases learning consolidation and facilitates synaptic plasticity through mechanisms dependent on NR2B subunits of the NMDA receptor.

    Science.gov (United States)

    Fontán-Lozano, Angela; Sáez-Cassanelli, José Luis; Inda, Mari Carmen; de los Santos-Arteaga, Mercedes; Sierra-Domínguez, Sergio Antonio; López-Lluch, Guillermo; Delgado-García, José María; Carrión, Angel Manuel

    2007-09-19

    One of the main focal points of aging research is the search for treatments that will prevent or ameliorate the learning and memory deficiencies associated with aging. Here we have examined the effects of maintaining mature mice on a long-term intermittent fasting diet (L-IFD). We found that L-IFD enhances learning and consolidation processes. We also assessed the long-term changes in synaptic efficiency in these animals. L-IFD mice showed an increase in low-theta-band oscillations, paired-pulse facilitation, and facilitation of long-term synaptic plasticity in the hippocampus with respect to mice fed ad libitum. In addition, we found an increase in the expression of the NMDA receptor subunit NR2B in some brain areas of L-IFD mice. Specific antagonism of this subunit in the hippocampus reversed the beneficial effects of L-IFD. These data provide a molecular and cellular mechanism by which L-IFD may enhance cognition, ameliorating some aging-associated cognitive deficits.

  4. Involvement of Gβγ subunits of Gi protein coupled with S1P receptor on multivesicular endosomes in F-actin formation and cargo sorting into exosomes.

    Science.gov (United States)

    Kajimoto, Taketoshi; Mohamed, Nesma Nabil Ibrahim; Badawy, Shaymaa Mohamed Mohamed; Matovelo, Shubi Ambwene; Hirase, Mitsuhiro; Nakamura, Shunsuke; Yoshida, Daisuke; Okada, Taro; Ijuin, Takeshi; Nakamura, Shun-Ichi

    2018-01-05

    Exosomes play a critical role in cell-to-cell communication by delivering cargo molecules to recipient cells. However, the mechanism underlying the generation of the exosomal multivesicular endosome (MVE) is one of the mysteries in the field of endosome research. Although sphingolipid metabolites such as ceramide and sphingosine 1-phosphate (S1P) are known to play important roles in MVE formation and maturation, the detailed molecular mechanisms are still unclear. Here, we show that Rho family GTPases, including Cdc42 and Rac1, are constitutively activated on exosomal MVEs and are regulated by S1P signaling as measured by fluorescence resonance energy transfer (FRET)-based conformational changes. Moreover, we detected S1P signaling-induced filamentous actin (F-actin) formation. A selective inhibitor of Gβγ subunits, M119, strongly inhibited both F-actin formation on MVEs and cargo sorting into exosomal intralumenal vesicles of MVEs, both of which were fully rescued by the simultaneous expression of constitutively active Cdc42 and Rac1. Our results shed light on the mechanism underlying exosomal MVE maturation and inform the understanding of the physiological relevance of continuous activation of the S1P receptor and subsequent downstream G protein signaling to Gβγ subunits/Rho family GTPases-regulated F-actin formation on MVEs for cargo sorting into exosomal intralumenal vesicles. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Decreased phosphorylation of δ and ε subunits of the acetylcholine receptor coincides with delayed postsynaptic maturation in PKC θ deficient mouse.

    Science.gov (United States)

    Lanuza, Maria A; Besalduch, Núria; González, Carmen; Santafé, Manel M; Garcia, Neus; Tomàs, Marta; Nelson, Phillip G; Tomàs, Josep

    2010-09-01

    Protein kinase C (PKC) activity is involved in the nicotinic acetylcholine receptor (nAChR) redistribution at the neuromuscular junction in vivo during postnatal maturation. Here we studied, in PKC theta (PKCtheta) deficient mice (KO), how the theta isoform of PKC is involved in the nAChR cluster maturation that is accompanied by the developmental activity-dependent neuromuscular synapse elimination process. We found that axonal elimination and dispersion of nAChR from the postsynaptic plaques and its redistribution to form the mature postsynaptic apparatus were delayed but not totally suppressed in PKCtheta deficient mice. Moreover, the delay in the maturation of the morphology of the nAChR clusters during the early postnatal synapse elimination period in the PKCtheta deficient mice coincides with a reduction in the PKCtheta-mediated phosphorylation on the delta subunit of the nAChR. In addition, we show evidence for PKCtheta regulation of PKA in normally phosphorylating the epsilon subunit of nAChR. We have also found that the theta isoform of PKC is located on the postsynaptic component of the neuromuscular junction but is also expressed by motoneurons in the spinal cord and in the motor nerve terminals. The results allow us to hypothesize that a spatially specific and opposing action of PKCtheta and PKA may result in activity-dependent alterations to synaptic connectivity at both the nerve inputs and the postsynaptic nAChR clusters. Copyright 2010 Elsevier Inc. All rights reserved.

  6. Interactions between the cyclic AMP receptor protein and the alpha subunit of RNA polymerase at the Escherichia coli galactose operon P1 promoter.

    Science.gov (United States)

    Attey, A; Belyaeva, T; Savery, N; Hoggett, J; Fujita, N; Ishihama, A; Busby, S

    1994-10-25

    DNAase I footprinting has been used to study open complexes between Escherichia coli RNA polymerase and the galactose operon P1 promoter, both in the absence and the presence of CRP (the cyclic AMP receptor protein, a transcription activator). From the effects of deletion of the C-terminal part of the RNA polymerase alpha subunit, we deduce that alpha binds at the upstream end of both the binary RNA polymerase-galP1 and ternary RNA polymerase-CRP-galP1 complexes. Disruption of the alpha-upstream contact suppresses open complex formation at galP1 at lower temperatures. In ternary RNA polymerase-CRP-galP1 complexes, alpha appears to make direct contact with Activating Region 1 in CRP. DNAase I footprinting has been used to detect and quantify interactions between purified alpha and CRP bound at galP1.

  7. Yeast two-hybrid screening of proteins interacting with plasmin receptor subunit: C-terminal fragment of annexin A2.

    Science.gov (United States)

    Li, Qun; Laumonnier, Yves; Syrovets, Tatiana; Simmet, Thomas

    2011-11-01

    To identify proteins that interact with the C-terminal fragment of annexin A2 (A2IC), generated by plasmin cleavage of the plasmin receptor, a heterotetramer (AA2t) containing annexin A2. The gene that encodes the A2IC fragment was obtained from PCR-amplified cDNA isolated from human monocytes, and was ligated into the pBTM116 vector using a DNA ligation kit. The resultant plasmid (pBTM116-A2IC) was sequenced with an ABI PRISM 310 Genetic Analyzer. The expression of an A2IC bait protein fused with a LexA-DNA binding domain (BD) was determined using Western blot analysis. The identification of proteins that interact with A2IC and are encoded in a human monocyte cDNA library was performed using yeast two-hybrid screening. The DNA sequences of the relevant cDNAs were determined using an ABI PRISM BigDye terminator cycle sequencing ready reaction kit. Nucleotide sequence databases were searched for homologous sequences using BLAST search analysis (http://www.ncbi.nlm.nih.gov). Confirmation of the interaction between the protein LexA-A2IC and each of cathepsin S and SNX17 was conducted using a small-scale yeast transformation and X-gal assay. The yeast transformed with plasmids encoding the bait proteins were screened with a human monocyte cDNA library by reconstituting full-length transcription factors containing the GAL4-active domain (GAL4-AD) as the prey in a yeast two-hybrid approach. After screening 1×10(7) clones, 23 independent β-Gal-positive clones were identified. Sequence analysis and a database search revealed that 15 of these positive clones matched eight different proteins (SNX17, ProCathepsin S, RPS2, ZBTB4, OGDH, CCDC32, PAPD4, and actin which was already known to interact with annexin A2). A2IC A2IC interacts with various proteins to form protein complexes, which may contribute to the molecular mechanism of monocyte activation induced by plasmin. The yeast two-hybrid system is an efficient approach for investigating protein interactions.

  8. Nicotinic acetylcholine receptors containing the α7-like subunit mediate contractions of muscles responsible for space positioning of the snail, Helix pomatia L. tentacle.

    Directory of Open Access Journals (Sweden)

    Tibor Kiss

    Full Text Available Three recently discovered tentacle muscles are crucial to perform patterned movements of upper tentacles of the terrestrial snail, Helix pomatia. The muscles receive central and peripheral excitatory cholinergic innervation lacking inhibitory innervation. Here, we investigate the pharmacology of acetylcholine (ACh responses in muscles to determine the properties of the ACh receptor (AChR, the functional availability of which was assessed using isotonic contraction measurement. Using broad spectrum of nicotinic and muscarinic ligands, we provide the evidence that contractions in the muscles are attributable to the activation of nAChRs that contain the α7-like subunit. Contractions could be evoked by nicotine, carbachol, succinylchloride, TMA, the selective α7-nAChR agonist choline chloride, 3-Bromocytisine and PNU-282987, and blocked by nAChR selective antagonists such as mytolon, hexamethonium, succinylchloride, d-tubocurarine, hemicholinium, DMDA (decamethonium, methyllycaconitine, α-Bungarotoxin (αBgTx and α-Conotoxin IMI. The specific muscarinic agonist oxotremorine and arecoline failed to elicit contractions. Based on these pharmacological properties we conclude that the Na+ and Ca2+ permeable AChRs of the flexor muscle are nicotinic receptors that contain the α7-like subunit. Immunodetection experiments confirmed the presence of α7- or α7-like AChRs in muscle cells, and α4-AChRs in nerves innervating the muscle. These results support the conclusion that the slowly desensitizing αBgTx-sensitive responses obtained from flexor muscles are produced by activation of α7- like AChRs. This is the first demonstration of postsynaptic expression and an obligatory role for a functional α7-like nAChR in the molluscan periphery.

  9. Lentviral-mediated RNAi to inhibit target gene expression of the porcine integrin αv subunit, the FMDV receptor, and against FMDV infection in PK-15 cells

    Directory of Open Access Journals (Sweden)

    Lin Tong

    2011-09-01

    Full Text Available Abstract Background shRNA targeting the integrin αv subunit, which is the foot-and-mouth disease virus (FMDV receptor, plays a key role in virus attachment to susceptible cells. We constructed a RNAi lentiviral vector, iαv pLenti6/BLOCK -iT™, which expressed siRNA targeting the FMDV receptor, the porcine integrin αv subunit, on PK-15 cells. We also produced a lentiviral stock, established an iαv-PK-15 cell line, evaluated the gene silencing efficiency of mRNA using real-time qRT-PCR, integrand αv expression by indirect immunofluorescence assay (IIF and cell enzyme linked immunosorbent assays (cell ELISA, and investigated the in vivo inhibitory effect of shRNA on FMDV replication in PK-15 cells. Results Our results indicated successful establishment of the iαv U6 RNAi entry vector and the iαv pLenti6/BLOCK -iT expression vector. The functional titer of obtained virus was 1.0 × 106 TU/mL. To compare with the control and mock group, the iαv-PK-15 group αv mRNA expression rate in group was reduced by 89.5%, whilst IIF and cell ELISA clearly indicated suppression in the experimental group. Thus, iαv-PK-15 cells could reduce virus growth by more than three-fold and there was a > 99% reduction in virus titer when cells were challenged with 102 TCID50 of FMDV. Conclusions Iαv-PK-15 cells were demonstrated as a cell model for anti-FMDV potency testing, and this study suggests that shRNA could be a viable therapeutic approach for controlling the severity of FMD infection and spread.

  10. Activation of the Hypoxia Inducible Factor 1α Subunit Pathway in Steatotic Liver Contributes to Formation of Cholesterol Gallstones.

    Science.gov (United States)

    Asai, Yoichiro; Yamada, Tetsuya; Tsukita, Sohei; Takahashi, Kei; Maekawa, Masamitsu; Honma, Midori; Ikeda, Masanori; Murakami, Keigo; Munakata, Yuichiro; Shirai, Yuta; Kodama, Shinjiro; Sugisawa, Takashi; Chiba, Yumiko; Kondo, Yasuteru; Kaneko, Keizo; Uno, Kenji; Sawada, Shojiro; Imai, Junta; Nakamura, Yasuhiro; Yamaguchi, Hiroaki; Tanaka, Kozo; Sasano, Hironobu; Mano, Nariyasu; Ueno, Yoshiyuki; Shimosegawa, Tooru; Katagiri, Hideki

    2017-05-01

    Hypoxia-inducible factor 1α subunit (HIF1A) is a transcription factor that controls the cellular response to hypoxia and is activated in hepatocytes of patients with nonalcoholic fatty liver disease (NAFLD). NAFLD increases the risk for cholesterol gallstone disease by unclear mechanisms. We studied the relationship between HIF1A and gallstone formation associated with liver steatosis. We performed studies with mice with inducible disruption of Hif1a in hepatocytes via a Cre adenoviral vector (inducible hepatocyte-selective HIF1A knockout [iH-HIFKO] mice), and mice without disruption of Hif1a (control mice). Mice were fed a diet rich in cholesterol and cholate for 1 or 2 weeks; gallbladders were collected and the number of gallstones was determined. Livers and biliary tissues were analyzed by histology, quantitative reverse-transcription polymerase chain reaction, immunohistochemistry, and immunoblots. We measured concentrations of bile acid, cholesterol, and phospholipid in bile and rates of bile flow. Primary hepatocytes and cholangiocytes were isolated and analyzed. HIF1A was knocked down in Hepa1-6 cells with small interfering RNAs. Liver biopsy samples from patients with NAFLD, with or without gallstones, were analyzed by quantitative reverse-transcription polymerase chain reaction. Control mice fed a diet rich in cholesterol and cholate developed liver steatosis with hypoxia; levels of HIF1A protein were increased in hepatocytes around central veins and 90% of mice developed cholesterol gallstones. Only 20% of the iH-HIFKO mice developed cholesterol gallstones. In iH-HIFKO mice, the biliary lipid concentration was reduced by 36%, compared with control mice, and bile flow was increased by 35%. We observed increased water secretion from hepatocytes into bile canaliculi to mediate these effects, resulting in suppression of cholelithogenesis. Hepatic expression of aquaporin 8 (AQP8) protein was 1.5-fold higher in iH-HIFKO mice than in control mice. Under hypoxic

  11. The heterotrimeric G protein Gβ1 interacts with the catalytic subunit of protein phosphatase 1 and modulates G protein-coupled receptor signaling in platelets.

    Science.gov (United States)

    Pradhan, Subhashree; Khatlani, Tanvir; Nairn, Angus C; Vijayan, K Vinod

    2017-08-11

    Thrombosis is caused by the activation of platelets at the site of ruptured atherosclerotic plaques. This activation involves engagement of G protein-coupled receptors (GPCR) on platelets that promote their aggregation. Although it is known that protein kinases and phosphatases modulate GPCR signaling, how serine/threonine phosphatases integrate with G protein signaling pathways is less understood. Because the subcellular localization and substrate specificity of the catalytic subunit of protein phosphatase 1 (PP1c) is dictated by PP1c-interacting proteins, here we sought to identify new PP1c interactors. GPCRs signal via the canonical heterotrimeric Gα and Gβγ subunits. Using a yeast two-hybrid screen, we discovered an interaction between PP1cα and the heterotrimeric G protein Gβ 1 subunit. Co-immunoprecipitation studies with epitope-tagged PP1c and Gβ 1 revealed that Gβ 1 interacts with the PP1c α, β, and γ1 isoforms. Purified PP1c bound to recombinant Gβ 1 -GST protein, and PP1c co-immunoprecipitated with Gβ 1 in unstimulated platelets. Thrombin stimulation of platelets induced the dissociation of the PP1c-Gβ 1 complex, which correlated with an association of PP1c with phospholipase C β3 (PLCβ3), along with a concomitant dephosphorylation of the inhibitory Ser 1105 residue in PLCβ3. siRNA-mediated depletion of GNB1 (encoding Gβ 1 ) in murine megakaryocytes reduced protease-activated receptor 4, activating peptide-induced soluble fibrinogen binding. Thrombin-induced aggregation was decreased in PP1cα -/- murine platelets and in human platelets treated with a small-molecule inhibitor of Gβγ. Finally, disruption of PP1c-Gβ 1 complexes with myristoylated Gβ 1 peptides containing the PP1c binding site moderately decreased thrombin-induced human platelet aggregation. These findings suggest that Gβ 1 protein enlists PP1c to modulate GPCR signaling in platelets.

  12. Mice with subtle reduction of NMDA NR1 receptor subunit expression have a selective decrease in mismatch negativity: Implications for schizophrenia prodromal population.

    Science.gov (United States)

    Featherstone, Robert E; Shin, Rick; Kogan, Jeffrey H; Liang, Yuling; Matsumoto, Mitsuyuki; Siegel, Steven J

    2015-01-01

    Reductions in glutamate function are regarded as an important contributory factor in schizophrenia. However, there is a paucity of animal models characterized by developmental and sustained reductions in glutamate function. Pharmacological models using NMDA antagonists have been widely used but these typically produce only transient changes in behavior and brain function. Likewise, mice with homozygous constitutive reductions in glutamate receptor expression show stable brain and behavioral changes, but many of these phenotypes are more severe than the human disease. The current study examines a variety of schizophrenia-related EEG measures in mice with a heterozygous alteration of the NMDA receptor NR1 subunit gene (NR1) that is known to result in reduced NR1 receptor expression in the homozygous mouse (NR1-/-). (NR1+/-) mice showed a 30% reduction in NR1 receptor expression and were reared after weaning in either group or isolated conditions. Outcome measures include the response to paired white noise stimuli, escalating inter-stimulus intervals (ISIs) and deviance-related mismatch negativity (MMN). In contrast to what has been reported in (NR1-/-) mice and mice treated with NMDA antagonists, (NR1+/-) mice showed no change on obligatory Event Related Potential (ERP) measures including the murine P50 and N100 equivalents (P20 and N40), or measures of baseline or evoked gamma power. Alternatively, (NR1+/-) mice showed a marked reduction in response to a deviant auditory tone during MMN task. Data suggest that EEG response to deviant, rather than static, stimuli may be more sensitive for detecting subtle changes in glutamate function. Deficits in these heterozygous NR1 knockdown mice are consistent with data demonstrating MMN deficits among family members of schizophrenia patients and among prodromal patients. Therefore, the current study suggests that (NR1+/-) mice may be among the most sensitive models for increased vulnerability to schizophrenia. Copyright

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

    Science.gov (United States)

    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…

  14. Zolpidem, a selective GABA(A) receptor alpha1 subunit agonist, induces comparable Fos expression in oxytocinergic neurons of the hypothalamic paraventricular and accessory but not supraoptic nuclei in the rat

    DEFF Research Database (Denmark)

    Kiss, Alexander; Søderman, Andreas; Bundzikova, Jana

    2006-01-01

    Functional activation of oxytocinergic (OXY) cells in the hypothalamic paraventricular (PVN), supraoptic (SON), and accessory (ACC) nuclei was investigated in response to acute treatment with Zolpidem (a GABA(A) receptor agonist with selectivity for alpha(1) subunits) utilizing dual Fos/OXY immun...

  15. Implementation of a fluorescence-based screening assay identifies histamine H3 receptor antagonists clobenpropit and iodophenpropit as subunit-selective N-methyl-D-aspartate receptor antagonists

    DEFF Research Database (Denmark)

    Hansen, Kasper Bø; Mullasseril, Praseeda; Dawit, Sara

    2010-01-01

    N-Methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels that mediate a slow, Ca(2+)-permeable component of excitatory synaptic transmission in the central nervous system and play a pivotal role in synaptic plasticity, neuronal development, and several neurological diseases. We describe...... a fluorescence-based assay that measures NMDA receptor-mediated changes in intracellular calcium in a BHK-21 cell line stably expressing NMDA receptor NR2D with NR1 under the control of a tetracycline-inducible promoter (Tet-On). The assay selectively identifies allosteric modulators by using supramaximal...

  16. CB2 cannabinoid receptors contribute to bacterial invasion and mortality in polymicrobial sepsis.

    Directory of Open Access Journals (Sweden)

    Balázs Csóka

    2009-07-01

    Full Text Available Sepsis is a major healthcare problem and current estimates suggest that the incidence of sepsis is approximately 750,000 annually. Sepsis is caused by an inability of the immune system to eliminate invading pathogens. It was recently proposed that endogenous mediators produced during sepsis can contribute to the immune dysfunction that is observed in sepsis. Endocannabinoids that are produced excessively in sepsis are potential factors leading to immune dysfunction, because they suppress immune cell function by binding to G-protein-coupled CB(2 receptors on immune cells. Here we examined the role of CB(2 receptors in regulating the host's response to sepsis.The role of CB(2 receptors was studied by subjecting CB(2 receptor wild-type and knockout mice to bacterial sepsis induced by cecal ligation and puncture. We report that CB(2 receptor inactivation by knockout decreases sepsis-induced mortality, and bacterial translocation into the bloodstream of septic animals. Furthermore, CB(2 receptor inactivation decreases kidney and muscle injury, suppresses splenic nuclear factor (NF-kappaB activation, and diminishes the production of IL-10, IL-6 and MIP-2. Finally, CB(2 receptor deficiency prevents apoptosis in lymphoid organs and augments the number of CD11b(+ and CD19(+ cells during CLP.Taken together, our results establish for the first time that CB(2 receptors are important contributors to septic immune dysfunction and mortality, indicating that CB(2 receptors may be therapeutically targeted for the benefit of patients suffering from sepsis.

  17. Receptor-binding domain of SARS-CoV spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine

    International Nuclear Information System (INIS)

    He Yuxian; Zhou Yusen; Liu Shuwen; Kou Zhihua; Li Wenhui; Farzan, Michael; Jiang Shibo

    2004-01-01

    The spike (S) protein of severe acute respiratory syndrome (SARS) coronavirus (CoV), a type I transmembrane envelope glycoprotein, consists of S1 and S2 domains responsible for virus binding and fusion, respectively. The S1 contains a receptor-binding domain (RBD) that can specifically bind to angiotensin-converting enzyme 2 (ACE2), the receptor on target cells. Here we show that a recombinant fusion protein (designated RBD-Fc) containing 193-amino acid RBD (residues 318-510) and a human IgG1 Fc fragment can induce highly potent antibody responses in the immunized rabbits. The antibodies recognized RBD on S1 domain and completely inhibited SARS-CoV infection at a serum dilution of 1:10,240. Rabbit antisera effectively blocked binding of S1, which contains RBD, to ACE2. This suggests that RBD can induce highly potent neutralizing antibody responses and has potential to be developed as an effective and safe subunit vaccine for prevention of SARS

  18. Essential role of NMDA receptor channel ε4 subunit (GluN2D in the effects of phencyclidine, but not methamphetamine.

    Directory of Open Access Journals (Sweden)

    Yoko Hagino

    Full Text Available Phencyclidine (PCP, a noncompetitive N-methyl-D-aspartate (NMDA receptor antagonist, increases locomotor activity in rodents and causes schizophrenia-like symptoms in humans. Although activation of the dopamine (DA pathway is hypothesized to mediate these effects of PCP, the precise mechanisms by which PCP induces its effects remain to be elucidated. The present study investigated the effect of PCP on extracellular levels of DA (DA(ex in the striatum and prefrontal cortex (PFC using in vivo microdialysis in mice lacking the NMDA receptor channel ε1 or ε4 subunit (GluRε1 [GluN2A] or GluRε4 [GluN2D] and locomotor activity. PCP significantly increased DA(ex in wildtype and GluRε1 knockout mice, but not in GluRε4 knockout mice, in the striatum and PFC. Acute and repeated administration of PCP did not increase locomotor activity in GluRε4 knockout mice. The present results suggest that PCP enhances dopaminergic transmission and increases locomotor activity by acting at GluRε4.

  19. Serotonin 2A receptors contribute to the regulation of risk-averse decisions

    DEFF Research Database (Denmark)

    Macoveanu, Julian; Rowe, James B; Hornboll, Bettina

    2013-01-01

    Pharmacological studies point to a role of the neurotransmitter serotonin (5-HT) in regulating the preference for risky decisions, yet the functional contribution of specific 5-HT receptors remains to be clarified. We used pharmacological fMRI to investigate the role of the 5-HT2A receptors...... in processing negative outcomes and regulating risk-averse behavior. During fMRI, twenty healthy volunteers performed a gambling task under two conditions: with or without blocking the 5-HT2A receptors. The volunteers repeatedly chose between small, likely rewards and large, unlikely rewards. Choices were...

  20. Antagonism at the NR2B subunit of NMDA receptors induces increased connectivity of the prefrontal and subcortical regions regulating reward behavior.

    Science.gov (United States)

    Gass, Natalia; Becker, Robert; Sack, Markus; Schwarz, Adam J; Reinwald, Jonathan; Cosa-Linan, Alejandro; Zheng, Lei; von Hohenberg, Christian Clemm; Inta, Dragos; Meyer-Lindenberg, Andreas; Weber-Fahr, Wolfgang; Gass, Peter; Sartorius, Alexander

    2018-04-01

    Evidence indicates that ketamine's rapid antidepressant efficacy likely results from its antagonism of NR2B-subunit-containing NMDA receptors (NMDAR). Since ketamine equally blocks NR2A- and NR2B-containing NMDAR, and has affinity to other receptors, NR2B-selective drugs might have improved therapeutic efficiency and side effect profile. We aimed to compare the effects of (S)-ketamine and two different types of NR2B-selective antagonists on functional brain networks in rats, in order to find common circuits, where their effects intersect, and that might explain their antidepressant action. The experimental design comprised four parallel groups of rats (N = 37), each receiving (S)-Ketamine, CP-101,606, Ro 25-6981 or saline. After compound injection, we acquired resting-state functional magnetic resonance imaging time series. We used graph theoretical approach to calculate brain network properties. Ketamine and CP-101,606 diminished the global clustering coefficient and small-worldness index. At the nodal level, all compounds induced increased connectivity of the regions mediating reward and cognitive aspects of emotional processing, such as ventromedial prefrontal cortex, septal nuclei, and nucleus accumbens. The dorsal hippocampus and regions involved in sensory processing and aversion, such as superior and inferior colliculi, exhibited an opposite effect. The effects common to ketamine and NR2B-selective compounds were localized to the same brain regions as those reported in depression, but in the opposite direction. The upregulation of the reward circuitry might partially underlie the antidepressant and anti-anhedonic effects of the antagonists and could potentially serve as a translational imaging phenotype for testing putative antidepressants, especially those targeting the NR2B receptor subtype.

  1. G Protein-Coupled Receptor-G-Protein βγ-Subunit Signaling Mediates Renal Dysfunction and Fibrosis in Heart Failure.

    Science.gov (United States)

    Kamal, Fadia A; Travers, Joshua G; Schafer, Allison E; Ma, Qing; Devarajan, Prasad; Blaxall, Burns C

    2017-01-01

    Development of CKD secondary to chronic heart failure (CHF), known as cardiorenal syndrome type 2 (CRS2), clinically associates with organ failure and reduced survival. Heart and kidney damage in CRS2 results predominantly from chronic stimulation of G protein-coupled receptors (GPCRs), including adrenergic and endothelin (ET) receptors, after elevated neurohormonal signaling of the sympathetic nervous system and the downstream ET system, respectively. Although we and others have shown that chronic GPCR stimulation and the consequent upregulated interaction between the G-protein βγ-subunit (Gβγ), GPCR-kinase 2, and β-arrestin are central to various cardiovascular diseases, the role of such alterations in kidney diseases remains largely unknown. We investigated the possible salutary effect of renal GPCR-Gβγ inhibition in CKD developed in a clinically relevant murine model of nonischemic hypertrophic CHF, transverse aortic constriction (TAC). By 12 weeks after TAC, mice developed CKD secondary to CHF associated with elevated renal GPCR-Gβγ signaling and ET system expression. Notably, systemic pharmacologic Gβγ inhibition by gallein, which we previously showed alleviates CHF in this model, attenuated these pathologic renal changes. To investigate a direct effect of gallein on the kidney, we used a bilateral ischemia-reperfusion AKI mouse model, in which gallein attenuated renal dysfunction, tissue damage, fibrosis, inflammation, and ET system activation. Furthermore, in vitro studies showed a key role for ET receptor-Gβγ signaling in pathologic fibroblast activation. Overall, our data support a direct role for GPCR-Gβγ in AKI and suggest GPCR-Gβγ inhibition as a novel therapeutic approach for treating CRS2 and AKI. Copyright © 2016 by the American Society of Nephrology.

  2. A three amino acid deletion in the transmembrane domain of the nicotinic acetylcholine receptor α6 subunit confers high-level resistance to spinosad in Plutella xylostella.

    Science.gov (United States)

    Wang, Jing; Wang, Xingliang; Lansdell, Stuart J; Zhang, Jianheng; Millar, Neil S; Wu, Yidong

    2016-04-01

    Spinosad is a macrocyclic lactone insecticide that acts primarily at the nicotinic acetylcholine receptors (nAChRs) of target insects. Here we describe evidence that high levels of resistance to spinosad in the diamondback moth (Plutella xylostella) are associated with a three amino acid (3-aa) deletion in the fourth transmembrane domain (TM4) of the nAChR α6 subunit (Pxα6). Following laboratory selection with spinosad, the SZ-SpinR strain of P. xylostella exhibited 940-fold resistance to spinosad. In addition, the selected insect population had 1060-fold cross-resistance to spinetoram but, in contrast, no cross-resistance to abamectin was observed. Genetic analysis indicates that spinosad resistance in SZ-SpinR is inherited as a recessive and autosomal trait, and that the 3-aa deletion (IIA) in TM4 of Pxα6 is tightly linked to spinosad resistance. Because of well-established difficulties in functional expression of cloned insect nAChRs, the analogous resistance-associated deletion mutation was introduced into a prototype nAChR (the cloned human α7 subunit). Two-electrode voltage-clamp recording with wild-type and mutated nAChRs expressed in Xenopus laevis oocytes indicated that the mutation causes a complete loss of agonist activation. In addition, radioligand binding studies indicated that the 3-aa deletion resulted in significantly lower-affinity binding of the extracellular neurotransmitter-binding site. These findings are consistent with the 3-amino acid (IIA) deletion within the transmembrane domain of Pxα6 being responsible for target-site resistance to spinosad in the SZ-SpinR strain of P. xylostella. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  3. Alternative-splicing in the exon-10 region of GABA(A receptor beta(2 subunit gene: relationships between novel isoforms and psychotic disorders.

    Directory of Open Access Journals (Sweden)

    Cunyou Zhao

    Full Text Available BACKGROUND: Non-coding single nucleotide polymorphisms (SNPs in GABRB2, the gene for beta(2-subunit of gamma-aminobutyric acid type A (GABA(A receptor, have been associated with schizophrenia (SCZ and quantitatively correlated to mRNA expression and alternative splicing. METHODS AND FINDINGS: Expression of the Exon 10 region of GABRB2 from minigene constructs revealed this region to be an "alternative splicing hotspot" that readily gave rise to differently spliced isoforms depending on intron sequences. This led to a search in human brain cDNA libraries, and the discovery of two novel isoforms, beta(2S1 and beta(2S2, bearing variations in the neighborhood of Exon-10. Quantitative real-time PCR analysis of postmortem brain samples showed increased beta(2S1 expression and decreased beta(2S2 expression in both SCZ and bipolar disorder (BPD compared to controls. Disease-control differences were significantly correlated with SNP rs187269 in BPD males for both beta(2S1 and beta(2S2 expressions, and significantly correlated with SNPs rs2546620 and rs187269 in SCZ males for beta(2S2 expression. Moreover, site-directed mutagenesis indicated that Thr(365, a potential phosphorylation site in Exon-10, played a key role in determining the time profile of the ATP-dependent electrophysiological current run-down. CONCLUSION: This study therefore provided experimental evidence for the importance of non-coding sequences in the Exon-10 region in GABRB2 with respect to beta(2-subunit splicing diversity and the etiologies of SCZ and BPD.

  4. Chronic MK-801 Application in Adolescence and Early Adulthood: A Spatial Working Memory Deficit in Adult Long-Evans Rats But No Changes in the Hippocampal NMDA Receptor Subunits

    Science.gov (United States)

    Uttl, Libor; Petrasek, Tomas; Sengul, Hilal; Svojanovska, Marketa; Lobellova, Veronika; Vales, Karel; Radostova, Dominika; Tsenov, Grygoriy; Kubova, Hana; Mikulecka, Anna; Svoboda, Jan; Stuchlik, Ales

    2018-01-01

    The role of NMDA receptors in learning, memory and hippocampal function has long been recognized. Post-mortem studies have indicated that the expression or subunit composition of the NMDA glutamate receptor subtype might be related to the impaired cognitive functions found in schizophrenia patients. NMDA receptor antagonists have been used to develop animal models of this disorder. There is accumulating evidence showing that not only the acute but also the chronic application of NMDA receptor antagonists may induce schizophrenia-like alterations in behavior and brain functions. However, limited evidence is available regarding the consequences of NMDA receptor blockage during periods of adolescence and early adulthood. This study tested the hypothesis that a 2-week treatment of male Long-Evans and Wistar rats with dizocilpine (MK-801; 0.5 mg/kg daily) starting at postnatal days (PD) 30 and 60 would cause a long-term cognitive deficit and changes in the levels of NMDA receptor subunits. The working memory version of the Morris water maze (MWM) and active place avoidance with reversal on a rotating arena (Carousel) requiring cognitive coordination and flexibility probed cognitive functions and an elevated-plus maze (EPM) was used to measure anxiety-like behavior. The western blot method was used to determine changes in NMDA receptor subunit levels in the hippocampus. Our results showed no significant changes in behaviors in Wistar rats. Slightly elevated anxiety-like behavior was observed in the EPM in Long-Evans rats with the onset of treatment on PD 30. Furthermore, Long-Evans rats treated from PD 60 displayed impaired working memory in the MWM. There were; however, no significant changes in the levels of NMDA receptor subunits because of MK-801 administration. These findings suggest that a 2-week treatment starting on PD 60 in Long-Evans rats leads to long-term changes in working memory, but this deficit is not paralleled by changes in NMDA receptor subunits. These

  5. Ketamine-induced inhibition of glycogen synthase kinase-3 contributes to the augmentation of AMPA receptor signaling

    Science.gov (United States)

    Beurel, Eléonore; Grieco, Steven F; Amadei, Celeste; Downey, Kimberlee; Jope, Richard S

    2016-01-01

    Objectives Sub-anesthetic doses of ketamine have been found to provide rapid antidepressant actions, indicating that the cellular signaling systems targeted by ketamine are potential sites for therapeutic intervention. Ketamine acts as an antagonist of N-methyl-D-aspartate (NMDA) receptors, and animal studies indicate that subsequent augmentation of signaling by α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors is critical for the antidepressant outcome. Methods In this study, we tested if the inhibitory effect of ketamine on glycogen synthase kinase-3 (GSK3) affected hippocampal cell-surface AMPA receptors using immunoblotting of membrane and synaptosomal extracts from wild-type and GSK3 knockin mice. Results Treatment with an antidepressant dose of ketamine increased the hippocampal membrane level of the AMPA glutamate receptor (GluA)1 subunit, but did not alter the localization of GluA2, GluA3, or GluA4. This effect of ketamine was abrogated in GSK3 knockin mice expressing mutant GSK3 that cannot be inhibited by ketamine, demonstrating that ketamine-induced inhibition of GSK3 is necessary for up-regulation of cell surface AMPA GluA1 subunits. AMPA receptor trafficking is regulated by post-synaptic density-95 (PSD-95), a substrate for GSK3. Ketamine treatment decreased the hippocampal membrane level of phosphorylated PSD-95 on Thr-19, the target of GSK3 that promotes AMPA receptor internalization. Conclusions These results demonstrate that ketamine-induced inhibition of GSK3 causes reduced phosphorylation of PSD-95, diminishing the internalization of AMPA GluA1 subunits to allow for augmented signaling through AMPA receptors following ketamine treatment. PMID:27687706

  6. Photoaffinity labeling of mammalian α1-adrenergic receptors: identification of the ligand binding subunit with a high affinity radioiodinated probe

    International Nuclear Information System (INIS)

    Leeb-Lundberg, L.M.F.; Dickinson, K.E.J.; Heald, S.L.

    1984-01-01

    A description is given of the synthesised and characterization of a novel high affinity radioiodinated α 1 -adrenergic receptor photoaffinity probe, 4-amino-6,7-dimethoxy-2-[4-[5-(4-azido-3-[ 125 I]iodophenyl)pentanoyl]-1-piperazinyl] quinazoline. In the absence of light, this ligand binds with high affinity (K/sub d/ = 130 pm) in a reverisble and saturable manner to sites in rat hepatic plasma membranes. The binding is stereoselective and competitively inhibited by adrenergic agonists and antagonists with an α 1 -adrenergic specificity. Upon photolysis, this ligand incorporates irreversibly into plasma membranes prepared from several mammalian tissues including rat liver, rat, guinea pig, and rabbit spleen, rabbit lung, and rabbit aorta vascular smooth muscle cells, also with typical α 1 -adrenergic specificity. Autoradiograms of such membrane samples subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveal a major specifically labeled polypeptide at M/sub 4/ = 78,000-85,000, depending on the tissue used, in addition to some lower molecular weight peptides. Protease inhibitors, in particular EDTA, a metalloprotease inhibitor, dramatically increases the predominance of the M/sub r/ = 78,000-85,000 polypeptide while attenuating the labeling of the lower molecular weight bands. This new high affinity radioiodinated photoaffinity probe should be of great value for the molecular characterization of the α 1 -adrenergic receptor

  7. High and low nightly running behavior associates with nucleus accumbens N-Methyl-d-aspartate receptor (NMDAR) NR1 subunit expression and NMDAR functional differences.

    Science.gov (United States)

    Grigsby, Kolter B; Kovarik, Cathleen M; Rottinghaus, George E; Booth, Frank W

    2018-04-03

    The extent to which N-Methyl-d-aspartate (NMDA) receptors facilitate the motivation to voluntarily wheel-run in rodents has yet to be determined. In so, we utilized female Wistar rats selectively bred to voluntarily run high (HVR) and low (LVR) nightly distances in order to examine if endogenous differences in nucleus accumbens (NAc) NMDA receptor expression and function underlies the propensity for high or low motivation to voluntarily wheel-run. 12-14 week old HVR and LVR females were used to examine: 1.) NAc mRNA and protein expression of NMDA subunits NR1, NR2A and NR2B; 2.) NMDA current responses in isolated medium spiny neurons (MSNs) and 3.) NMDA-evoked dopamine release in an ex vivo preparation of NAc punches. Expectedly, there was a large divergence in nightly running distance and time between HVR and LVR rats. We saw a significantly higher mRNA and protein expression of NR1 in HVR compared to LVR rats, while seeing no difference in the expression of NR2A or NR2B. There was a greater current response to a 500 ms application of 300 μM of NMDA in medium-spiny neurons isolated from the NAc HVR compared to LVR animals. On average, NMDA-evoked punches (50 μM of NMDA for 10 min) taken from HVR rats retained ∼54% of the dopamine content compared to their bilateral non-evoked sides, while evoked punches from LVR animals showed no statistical decrease in dopamine content compared to their non-evoked sides. Collectively, these data suggest a potential link between NAc NR1 subunit expression as well as NMDA function and the predisposition for nightly voluntary running behavior in rats. In light of the epidemic rise in physical inactivity, these findings have the potential to explain a neuro-molecular mechanism that regulates the motivation to be physically active. Copyright © 2018 Elsevier B.V. All rights reserved.

  8. 5-HT1A receptor blockade reverses GABA(A) receptor alpha(3) subunit-mediated anxiolytic effects on stress-induced hyperthermia

    NARCIS (Netherlands)

    Vinkers, Christiaan H.; van Oorschot, Ruud; Korte, S. Mechiel; Olivier, Berend; Groenink, Lucianne

    Stress-related disorders are associated with dysfunction of both serotonergic and GABAergic pathways, and clinically effective anxiolytics act via both neurotransmitter systems. As there is evidence that the GABA(A) and the serotonin receptor system interact, a serotonergic component in the

  9. Correlated cone noise decreases rod signal contributions to the post-receptoral pathways.

    Science.gov (United States)

    Hathibelagal, Amithavikram R; Feigl, Beatrix; Zele, Andrew J

    2018-04-01

    This study investigated how invisible extrinsic temporal white noise that correlates with the activity of one of the three [magnocellular (MC), parvocellular (PC), or koniocellular (KC)] post-receptoral pathways alters mesopic rod signaling. A four-primary photostimulator provided independent control of the rod and three cone photoreceptor excitations. The rod contributions to the three post-receptoral pathways were estimated by perceptually matching a 20% contrast rod pulse by independently varying the LMS (MC pathway), +L-M (PC pathway), and S-cone (KC pathway) excitations. We show that extrinsic cone noise caused a predominant decrease in the overall magnitude and ratio of the rod contributions to each pathway. Thus, the relative cone activity in the post-receptoral pathways determines the relative mesopic rod inputs to each pathway.

  10. Nature and regulation of the insulin receptor: structure and function

    International Nuclear Information System (INIS)

    Czech, M.P.

    1985-01-01

    Native, cell-surface insulin receptor consists of two glycoprotein subunit types with apparent masses of about 125,000 daltons (alpha subunit) and 90,000 daltons (beta subunit). The alpha and beta insulin-receptor subunits seem to have distinct functions such that alpha appears to bind hormone whereas beta appears to possess intrinsic tyrosine kinase activity. In detergent extracts, insulin activates receptor autophosphorylation of tyrosine residues on its beta subunit, whereas in the presence of reductant, the alpha subunit is also phosphorylated. In intact cells, insulin activates serine/threonine phosphorylation of insulin receptor beta subunit as well as tyrosine phosphorylation. The biological role of the receptor-associated tyrosine kinase is not known. The insulin receptor kinase is regulated by beta-adrenergic agonists and other agents that elevate cAMP in adipocytes, presumably via the cAMP-dependent protein kinase. Such agents decrease receptor affinity for insulin and partially uncouple receptor tyrosine kinase activity from activation by insulin. These effects appear to contribute to the biological antagonism between insulin and beta-agonists. These data suggest the hypothesis that a complex network of tyrosine and serine/threonine phosphorylations on the insulin receptor modulate its binding and kinase activities in an antagonistic manner

  11. Piroxicam inhibits NMDA receptor-mediated excitotoxicity through allosteric inhibition of the GluN2B subunit: an in silico study elucidating a novel mechanism of action of the drug.

    Science.gov (United States)

    Mazumder, Muhammed Khairujjaman; Borah, Anupom

    2014-12-01

    Hyperactivation of GluN2B subunit containing N-methyl-d-aspartate receptors (NMDARs) significantly contributes to the development of several neurodegenerative diseases through a process called excitotoxicity. NMDARs are voltage-gated Ca2+ channels which when activated lead to excessive influx of Ca2+ into neurons thereby exacerbating several calcium-dependent pathways that cause oxidative stress and apoptosis. Several drugs are presently in use to counter the NMDAR-mediated excitotoxic events among which Ifenprodil and its derivatives are GluN2B selective allosteric antagonists. Certain non-steroidal anti-inflammatory drugs (NSAIDs) have also been reported to inhibit NMDARs and the resultant pathologies. Meanwhile, Piroxicam, which is a NSAID, has been reported to be protective in cerebral ischemia-induced neurodegeneration through various pathways. Since Piroxicam has more number of interacting groups as compared to other NSAIDs and also has structural similarities with Ifenprodil, we thought it prudent that Piroxicam may inhibit NMDARs similar to Ifenprodil. By using molecular docking as a tool, we validated the hypothesis and hereby report for the first time that Piroxicam can inhibit GluN2B containing NMDARs through allosteric mode similar to the well known selective antagonist--Ifenprodil; and thus can be a therapeutic drug for the prevention of excitotoxic neurodegeneration. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. α7 and β2 Nicotinic Acetylcholine Receptor Subunits Form Heteromeric Receptor Complexes that Are Expressed in the Human Cortex and Display Distinct Pharmacological Properties

    DEFF Research Database (Denmark)

    Thomsen, Morten Skøtt; Zwart, Ruud; Ursu, Daniel

    2015-01-01

    The existence of α7β2 nicotinic acetylcholine receptors (nAChRs) has recently been demonstrated in both the rodent and human brain. Since α7-containing nAChRs are promising drug targets for schizophrenia and Alzheimer's disease, it is critical to determine whether α7β2 nAChRs are present in the h......The existence of α7β2 nicotinic acetylcholine receptors (nAChRs) has recently been demonstrated in both the rodent and human brain. Since α7-containing nAChRs are promising drug targets for schizophrenia and Alzheimer's disease, it is critical to determine whether α7β2 nAChRs are present...

  13. Transcriptional regulators of Na, K-ATPase subunits

    Directory of Open Access Journals (Sweden)

    Zhiqin eLi

    2015-10-01

    Full Text Available The Na,K-ATPase classically serves as an ion pump creating an electrochemical gradient across the plasma membrane that is essential for transepithelial transport, nutrient uptake and membrane potential. In addition, Na,K-ATPase also functions as a receptor, a signal transducer and a cell adhesion molecule. With such diverse roles, it is understandable that the Na,K-ATPase subunits, the catalytic alpha-subunit, the beta-subunit and the FXYD proteins, are controlled extensively during development and to accommodate physiological needs. The spatial and temporal expression of Na,K-ATPase is partially regulated at the transcriptional level. Numerous transcription factors, hormones, growth factors, lipids and extracellular stimuli modulate the transcription of the Na,K-ATPase subunits. Moreover, epigenetic mechanisms also contribute to the regulation of Na,K-ATPase expression. With the ever growing knowledge about diseases associated with the malfunction of Na,K-ATPase, this review aims at summarizing the best-characterized transcription regulators that modulate Na,K-ATPase subunit levels. As abnormal expression of Na,K-ATPase subunits have been observed in many carcinoma, we will also discuss transcription factors that are associated with epithelial-to-mesenchymal transition, a crucial step in the progression of many tumors to malignant disease.

  14. Kinin B1 receptors contributes to acute pain following minor surgery in humans

    Directory of Open Access Journals (Sweden)

    Brahim Jaime S

    2010-02-01

    early phase of inflammation and inflammatory pain. The up-regulation of B1 receptors may contribute to acute inflammatory pain through TRPV1 activation.

  15. The number and distribution of AMPA receptor channels containing fast kinetic GluA3 and GluA4 subunits at auditory nerve synapses depend on the target cells.

    Science.gov (United States)

    Rubio, María E; Matsui, Ko; Fukazawa, Yugo; Kamasawa, Naomi; Harada, Harumi; Itakura, Makoto; Molnár, Elek; Abe, Manabu; Sakimura, Kenji; Shigemoto, Ryuichi

    2017-11-01

    The neurotransmitter receptor subtype, number, density, and distribution relative to the location of transmitter release sites are key determinants of signal transmission. AMPA-type ionotropic glutamate receptors (AMPARs) containing GluA3 and GluA4 subunits are prominently expressed in subsets of neurons capable of firing action potentials at high frequencies, such as auditory relay neurons. The auditory nerve (AN) forms glutamatergic synapses on two types of relay neurons, bushy cells (BCs) and fusiform cells (FCs) of the cochlear nucleus. AN-BC and AN-FC synapses have distinct kinetics; thus, we investigated whether the number, density, and localization of GluA3 and GluA4 subunits in these synapses are differentially organized using quantitative freeze-fracture replica immunogold labeling. We identify a positive correlation between the number of AMPARs and the size of AN-BC and AN-FC synapses. Both types of AN synapses have similar numbers of AMPARs; however, the AN-BC have a higher density of AMPARs than AN-FC synapses, because the AN-BC synapses are smaller. A higher number and density of GluA3 subunits are observed at AN-BC synapses, whereas a higher number and density of GluA4 subunits are observed at AN-FC synapses. The intrasynaptic distribution of immunogold labeling revealed that AMPAR subunits, particularly GluA3, are concentrated at the center of the AN-BC synapses. The central distribution of AMPARs is absent in GluA3-knockout mice, and gold particles are evenly distributed along the postsynaptic density. GluA4 gold labeling was homogenously distributed along both synapse types. Thus, GluA3 and GluA4 subunits are distributed at AN synapses in a target-cell-dependent manner.

  16. Nuclear respiratory factor 2 regulates the expression of the same NMDA receptor subunit genes as NRF-1: both factors act by a concurrent and parallel mechanism to couple energy metabolism and synaptic transmission.

    Science.gov (United States)

    Priya, Anusha; Johar, Kaid; Wong-Riley, Margaret T T

    2013-01-01

    Neuronal activity and energy metabolism are tightly coupled processes. Previously, we found that nuclear respiratory factor 1 (NRF-1) transcriptionally co-regulates energy metabolism and neuronal activity by regulating all 13 subunits of the critical energy generating enzyme, cytochrome c oxidase (COX), as well as N-methyl-d-aspartate (NMDA) receptor subunits 1 and 2B, GluN1 (Grin1) and GluN2B (Grin2b). We also found that another transcription factor, nuclear respiratory factor 2 (NRF-2 or GA-binding protein) regulates all subunits of COX as well. The goal of the present study was to test our hypothesis that NRF-2 also regulates specific subunits of NMDA receptors, and that it functions with NRF-1 via one of three mechanisms: complementary, concurrent and parallel, or a combination of complementary and concurrent/parallel. By means of multiple approaches, including in silico analysis, electrophoretic mobility shift and supershift assays, in vivo chromatin immunoprecipitation of mouse neuroblastoma cells and rat visual cortical tissue, promoter mutations, real-time quantitative PCR, and western blot analysis, NRF-2 was found to functionally regulate Grin1 and Grin2b genes, but not any other NMDA subunit genes. Grin1 and Grin2b transcripts were up-regulated by depolarizing KCl, but silencing of NRF-2 prevented this up-regulation. On the other hand, over-expression of NRF-2 rescued the down-regulation of these subunits by the impulse blocker TTX. NRF-2 binding sites on Grin1 and Grin2b are conserved among species. Our data indicate that NRF-2 and NRF-1 operate in a concurrent and parallel manner in mediating the tight coupling between energy metabolism and neuronal activity at the molecular level. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. The association between the nicotinic acetylcholine receptor α4 subunit gene (CHRNA4 rs1044396 and Internet gaming disorder in Korean male adults.

    Directory of Open Access Journals (Sweden)

    Jo-Eun Jeong

    Full Text Available The primary aim of this study was to investigate the genetic predisposition of Internet gaming disorder (IGD, and the secondary aim was to compare the results to those of alcohol dependence (AD. Two independent case-control studies were conducted. A total of 30 male participants with IGD, diagnosed according to the 5th edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5 criteria, and 30 sex-matched controls participated in study 1. We designed targeted exome sequencing (TES to test for 72 candidate genes that have been implicated in the pathogenesis of addiction. The genes included seven neurotransmitter (dopamine, serotonin, glutamate, r-aminobutyric acid (GABA, norepinephrine, acetylcholine, and opioid system genes. A total of 31 male in-patients with AD and 29 normal male controls (NC were enrolled in study 2. The same 72 genes included in study 1 and ten additional genes related to alcohol-metabolic enzyme were selected as the target genes, and we identified the genetic variants using the same method (TES. The IGD group had a lower frequency of the T allele of rs1044396 in the nicotinic acetylcholine receptor alpha 4 subunit (CHRNA4, and this variant represents a protective allele against IGD. However, we did not find a significant difference in the polymorphisms of the 72 genes that encode neurotransmitter systems between the AD and NC groups. This study demonstrated that rs1044396 of CHRNA4 was significantly associated with IGD.

  18. Rapid PCR-mediated synthesis of competitor molecules for accurate quantification of beta(2) GABA(A) receptor subunit mRNA.

    Science.gov (United States)

    Vela, J; Vitorica, J; Ruano, D

    2001-12-01

    We describe a fast and easy method for the synthesis of competitor molecules based on non-specific conditions of PCR. RT-competitive PCR is a sensitive technique that allows quantification of very low quantities of mRNA molecules in small tissue samples. This technique is based on the competition established between the native and standard templates for nucleotides, primers or other factors during PCR. Thus, the most critical parameter is the use of good internal standards to generate a standard curve from which the amount of native sequences can be properly estimated. At the present time different types of internal standards and methods for their synthesis have been described. Normally, most of these methods are time-consuming and require the use of different sets of primers, different rounds of PCR or specific modifications, such as site-directed mutagenesis, that need subsequent analysis of the PCR products. Using our method, we obtained in a single round of PCR and with the same primer pair, competitor molecules that were successfully used in RT-competitive PCR experiments. The principal advantage of this method is high versatility and economy. Theoretically it is possible to synthesize a specific competitor molecule for each primer pair used. Finally, using this method we have been able to quantify the increase in the expression of the beta(2) GABA(A) receptor subunit mRNA that occurs during rat hippocampus development.

  19. Bacopa monnieri Extract (CDRI-08 Modulates the NMDA Receptor Subunits and nNOS-Apoptosis Axis in Cerebellum of Hepatic Encephalopathy Rats

    Directory of Open Access Journals (Sweden)

    Papia Mondal

    2015-01-01

    Full Text Available Hepatic encephalopathy (HE, characterized by impaired cerebellar functions during chronic liver failure (CLF, involves N-methyl-D-aspartate receptor (NMDAR overactivation in the brain cells. Bacopa monnieri (BM extract is a known neuroprotectant. The present paper evaluates whether BM extract is able to modulate the two NMDAR subunits (NR2A and NR2B and its downstream mediators in cerebellum of rats with chronic liver failure (CLF, induced by administration of 50 mg/kg bw thioacetamide (TAA i.p. for 14 days, and in the TAA group rats orally treated with 200 mg/kg bw BM extract from days 8 to 14. NR2A is known to impart neuroprotection and that of NR2B induces neuronal death during NMDAR activation. Neuronal nitric oxide synthase- (nNOS- apoptosis pathway is known to mediate NMDAR led excitotoxicity. The level of NR2A was found to be significantly reduced with a concomitant increase of NR2B in cerebellum of the CLF rats. This was consistent with significantly enhanced nNOS expression, nitric oxide level, and reduced Bcl2/Bax ratio. Moreover, treatment with BM extract reversed the NR2A/NR2B ratio and also normalized the levels of nNOS-apoptotic factors in cerebellum of those rats. The findings suggest modulation of NR2A and NR2B expression by BM extract to prevent neurochemical alterations associated with HE.

  20. PRDM16 enhances nuclear receptor-dependent transcription of the brown fat-specific Ucp1 gene through interactions with Mediator subunit MED1.

    Science.gov (United States)

    Iida, Satoshi; Chen, Wei; Nakadai, Tomoyoshi; Ohkuma, Yoshiaki; Roeder, Robert G

    2015-02-01

    PR domain-containing 16 (PRDM16) induces expression of brown fat-specific genes in brown and beige adipocytes, although the underlying transcription-related mechanisms remain largely unknown. Here, in vitro studies show that PRDM16, through its zinc finger domains, directly interacts with the MED1 subunit of the Mediator complex, is recruited to the enhancer of the brown fat-specific uncoupling protein 1 (Ucp1) gene through this interaction, and enhances thyroid hormone receptor (TR)-driven transcription in a biochemically defined system in a Mediator-dependent manner, thus providing a direct link to the general transcription machinery. Complementary cell-based studies show that upon forskolin treatment, PRDM16 induces Ucp1 expression in undifferentiated murine embryonic fibroblasts, that this induction depends on MED1 and TR, and, consistent with a direct effect, that PRDM16 is recruited to the Ucp1 enhancer. Related studies have defined MED1 and PRDM16 interaction domains important for Ucp1 versus Ppargc1a induction by PRDM16. These results reveal novel mechanisms for PRDM16 function through the Mediator complex. © 2015 Iida et al.; Published by Cold Spring Harbor Laboratory Press.

  1. Identification of the ligand-binding subunit of the human 5-hydroxytryptamine1A receptor with N-(p-azido-m-[125I] iodophenethyl)spiperone, a high affinity radioiodinated photoaffinity probe

    International Nuclear Information System (INIS)

    Raymond, J.R.; Fargin, A.; Lohse, M.J.; Regan, J.W.; Senogles, S.E.; Lefkowitz, R.J.; Caron, M.G.

    1989-01-01

    The ligand-binding subunit of the human 5-hydroxytryptamine1A (5-HT1A) receptor transiently expressed in COS-7 cells and of the native human 5-HT1A receptor derived from hippocampus and frontal cortex were identified by photoaffinity labeling with N-(p-azido-m-[125I]iodophenethyl)spiperone [( 125I]N3-NAPS), previously characterized as a high affinity radioiodinated D2-dopamine receptor probe. The identity of the ligand-binding subunit was confirmed by immunoprecipitation with an antipeptide rabbit antiserum, JWR21, raised against a synthetic peptide derived from the predicted amino acid sequence of the putative third intracellular loop of the human 5-HT1A receptor. In transiently transfected COS-7 cells expressing 14 +/- 3 pmol/mg of protein human 5-HT1A receptors, a single broad 75-kDa band was photoaffinity labeled by [125I]N3-NAPS. This band displayed the expected pharmacology of the 5-HT1A receptor, as evidenced by the ability of a series of competing ligands to block [125I]N3-NAPS photoincorporation. Moreover, antiserum JWR21 specifically and quantitatively immunoprecipitated the 75-kDa photoaffinity-labeled band from a soluble extract of the transfected COS-7 cell membranes, further confirming its identity. Finally, utilizing a combination of photoaffinity labeling and immunoprecipitation, the native ligand-binding subunit of 62-64 kDa was identified in human hippocampus and frontal cortex. The availability of the high specific activity, high affinity, photoaffinity ligand [125I]N3-NAPS and of a potent immunoprecipitating antiserum (JWR21) should greatly facilitate the biochemical characterization of the human 5-HT1A receptor

  2. A new splice variant of the major subunit of human asialoglycoprotein receptor encodes a secreted form in hepatocytes.

    Directory of Open Access Journals (Sweden)

    Jia Liu

    Full Text Available BACKGROUND: The human asialoglycoprotein receptor (ASGPR is composed of two polypeptides, designated H1 and H2. While variants of H2 have been known for decades, the existence of H1 variants has never been reported. PRINCIPAL FINDINGS: We identified two splice variants of ASGPR H1 transcripts, designated H1a and H1b, in human liver tissues and hepatoma cells. Molecular cloning of ASGPR H1 variants revealed that they differ by a 117 nucleotide segment corresponding to exon 2 in the ASGPR genomic sequence. Thus, ASGPR variant H1b transcript encodes a protein lacking the transmembrane domain. Using an H1b-specific antibody, H1b protein and a functional soluble ASGPR (sASGPR composed of H1b and H2 in human sera and in hepatoma cell culture supernatant were identified. The expression of ASGPR H1a and H1b in Hela cells demonstrated the different cellular loctions of H1a and H1b proteins at cellular membranes and in intracellular compartments, respectively. In vitro binding assays using fluorescence-labeled sASGPR or the substract ASOR revealed that the presence of sASGPR reduced the binding of ASOR to cells. However, ASOR itself was able to enhance the binding of sASGPR to cells expressing membrane-bound ASGPR. Further, H1b expression is reduced in liver tissues from patients with viral hepatitis. CONCLUSIONS: We conclude that two naturally occurring ASGPR H1 splice variants are produced in human hepatocytes. A hetero-oligomeric complex sASGPR consists of the secreted form of H1 and H2 and may bind to free substrates in circulation and carry them to liver tissue for uptake by ASGPR-expressing hepatocytes.

  3. Identification of a new adapter protein that may link the common beta subunit of the receptor for granulocyte/macrophage colony-stimulating factor, interleukin (IL)-3, and IL-5 to phosphatidylinositol 3-kinase.

    Science.gov (United States)

    Jücker, M; Feldman, R A

    1995-11-17

    Binding of human granulocyte/macrophage colony-stimulating factor (hGM-CSF) to its receptor induces the rapid activation of phosphatidylinositol-3 kinase (PI 3-kinase). As hGM-CSF receptor (hGMR) does not contain a consensus sequence for binding of PI 3-kinase, hGMR must use a distinct mechanism for its association with and activation of PI 3-kinase. Here, we describe the identification of a tyrosine-phosphorylated protein of 76-85 kDa (p80) that associates with the common beta subunit of hGMR and with the SH2 domains of the p85 subunit of PI 3-kinase in hGM-CSF-stimulated cells. Src/Yes and Lyn were tightly associated with the p80.PI 3-kinase complex, suggesting that p80 and other phosphotyrosyl proteins present in the complex were phosphorylated by Src family kinases. Tyrosine phosphorylation of p80 was only detected in hGM-CSF or human interleukin-3-stimulated cells, suggesting that activation of p80 might be specific for signaling via the common beta subunit. We postulate that p80 functions as an adapter protein that may participate in linking the hGM-CSF receptor to the PI 3-kinase signaling pathway.

  4. Bicarbonate Contributes to GABAA Receptor-Mediated Neuronal Excitation in Surgically-Resected Human Hypothalamic Hamartomas

    Science.gov (United States)

    Do-Young, Kim; Fenoglio, Kristina A.; Kerrigan, John F.; Rho, Jong M.

    2009-01-01

    SUMMARY The role of bicarbonate (HCO3-) in GABAA receptor-mediated depolarization of human hypothalamic hamartoma (HH) neurons was investigated using cellular electrophysiological and calcium imaging techniques. Activation of GABAA receptors with muscimol (30 μM) provoked neuronal excitation in over 70% of large (18-22 μM) HH neurons in HCO3- buffer. Subsequent perfusion of HCO3--free HEPES buffer produced partial suppression of muscimol-induced excitation. Additionally, 53% of large HH neurons under HCO3--free conditions exhibited reduced intracellular calcium accumulation by muscimol. These results suggest that HCO3- efflux through GABAA receptors on a subpopulation of large HH neurons may contribute to membrane depolarization and subsequent activation of L-type calcium channels. PMID:19022626

  5. A highly conserved glycine within linker I and the extreme C terminus of G protein alpha subunits interact cooperatively in switching G protein-coupled receptor-to-effector specificity

    DEFF Research Database (Denmark)

    Kostenis, Evi; Martini, Lene; Ellis, James

    2004-01-01

    Numerous studies have attested to the importance of the extreme C terminus of G protein alpha subunits in determining their selectivity of receptor recognition. We have previously reported that a highly conserved glycine residue within linker I is important for constraining the fidelity of receptor...... recognition by Galpha(q) proteins. Herein, we explored whether both modules (linker I and extreme C terminus) interact cooperatively in switching G protein-coupled receptor (GPCR)-to-effector specificity and created as models mutant Galpha(q) proteins in which glycine was replaced with various amino acids...... and the C-terminal five Galpha(q) residues with the corresponding Galpha(i) or Galpha(s) sequence. Coupling properties of the mutated Galpha(q) proteins were determined after coexpression with a panel of 13 G(i)-and G(s) -selective receptors and compared with those of Galpha proteins modified in only one...

  6. Endothelin B receptors contribute to retinal ganglion cell loss in a rat model of glaucoma.

    Directory of Open Access Journals (Sweden)

    Alena Z Minton

    Full Text Available Glaucoma is an optic neuropathy, commonly associated with elevated intraocular pressure (IOP characterized by optic nerve degeneration, cupping of the optic disc, and loss of retinal ganglion cells which could lead to loss of vision. Endothelin-1 (ET-1 is a 21-amino acid vasoactive peptide that plays a key role in the pathogenesis of glaucoma; however, the receptors mediating these effects have not been defined. In the current study, endothelin B (ET(B receptor expression was assessed in vivo, in the Morrison's ocular hypertension model of glaucoma in rats. Elevation of IOP in Brown Norway rats produced increased expression of ET(B receptors in the retina, mainly in retinal ganglion cells (RGCs, nerve fiber layer (NFL, and also in the inner plexiform layer (IPL and inner nuclear layer (INL. To determine the role of ET(B receptors in neurodegeneration, Wistar-Kyoto wild type (WT and ET(B receptor-deficient (KO rats were subjected to retrograde labeling with Fluoro-Gold (FG, following which IOP was elevated in one eye while the contralateral eye served as control. IOP elevation for 4 weeks in WT rats caused an appreciable loss of RGCs, which was significantly attenuated in KO rats. In addition, degenerative changes in the optic nerve were greatly reduced in KO rats compared to those in WT rats. Taken together, elevated intraocular pressure mediated increase in ET(B receptor expression and its activation may contribute to a decrease in RGC survival as seen in glaucoma. These findings raise the possibility of using endothelin receptor antagonists as neuroprotective agents for the treatment of glaucoma.

  7. Activation of neurokinin-1 receptors during ozone inhalation contributes to epithelial injury and repair.

    Science.gov (United States)

    Oslund, Karen L; Hyde, Dallas M; Putney, Leialoha F; Alfaro, Mario F; Walby, William F; Tyler, Nancy K; Schelegle, Edward S

    2008-09-01

    We investigated the importance of neurokinin (NK)-1 receptors in epithelial injury and repair and neutrophil function. Conscious Wistar rats were exposed to 1 ppm ozone or filtered air for 8 hours, followed by an 8-hour postexposure period. Before exposure, we administered either the NK-1 receptor antagonist, SR140333, or saline as a control. Ethidium homodimer was instilled into lungs as a marker of necrotic airway epithelial cells. After fixation, whole mounts of airway dissected lung lobes were immunostained for 5-bromo-2'-deoxyuridine, a marker of epithelial proliferation. Both ethidium homodimer and 5-bromo-2'-deoxyuridine-positive epithelial cells were quantified in specific airway generations. Rats treated with the NK-1 receptor antagonist had significantly reduced epithelial injury and epithelial proliferation compared with control rats. Sections of terminal bronchioles showed no significant difference in the number of neutrophils in airways between groups. In addition, staining ozone-exposed lung sections for active caspase 3 showed no apoptotic cells, but ethidium-positive cells colocalized with the orphan nuclear receptor, Nur77, a marker of nonapoptotic, programmed cell death mediated by the NK-1 receptor. An immortalized human airway epithelial cell line, human bronchial epithelial-1, showed no significant difference in the number of oxidant stress-positive cells during exposure to hydrogen peroxide and a range of SR140333 doses, demonstrating no antioxidant effect of the receptor antagonist. We conclude that activation of the NK-1 receptor during acute ozone inhalation contributes to epithelial injury and subsequent epithelial proliferation, a critical component of repair, but does not influence neutrophil emigration into airways.

  8. Contribution of 5-HT2A receptors on diaphragmatic recovery after chronic cervical spinal cord injury.

    Science.gov (United States)

    Lee, Kun-Ze; Gonzalez-Rothi, Elisa J

    2017-10-01

    Unilateral C2 spinal cord hemisection (C2Hx) interrupts bulbospinal respiratory pathways innervating ipsilateral phrenic motoneurons, resulting in cessation of ipsilateral diaphragm motor output. Plasticity within the spinal neural circuitry controlling the diaphragm can induce partial recovery of phrenic bursting which correlates with the time-dependent return of spinal serotonin (5-HT) immunoreactivity in the vicinity of phrenic motoneurons. The 5-HT 2A receptor subtype is present on phrenic motoneurons and its expression is up-regulated after cervical spinal cord injury; however the functional role of these receptors following injury has not been clearly defined. The present study evaluated the functional role of 5-HT 2A receptors by testing the hypothesis that pharmacologic blockade would attenuate diaphragm activity in rats with chronic cervical spinal cord injury. Bilateral diaphragm electromyography (EMG) was performed in vagal-intact and spontaneously breathing rats before and after intravenous administration of the 5-HT 2A receptor antagonist Ketanserin (1mg/kg). Intravenous ketanserin significantly attenuated ipsilateral diaphragm EMG activity in C2Hx animals but had no impact on diaphragm output in uninjured animals. We conclude that 5-HT 2A receptor activation contributes to the recovery of ipsilateral phrenic motor output after chronic cervical spinal cord injury. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. C-Type Lectin-Like Receptor 2 Suppresses AKT Signaling and Invasive Activities of Gastric Cancer Cells by Blocking Expression of Phosphoinositide 3-Kinase Subunits.

    Science.gov (United States)

    Wang, Lan; Yin, Jie; Wang, Xuefei; Shao, Miaomiao; Duan, Fangfang; Wu, Weicheng; Peng, Peike; Jin, Jing; Tang, Yue; Ruan, Yuanyuan; Sun, Yihong; Gu, Jianxin

    2016-05-01

    C-type lectin-like receptor 2 (CLEC2) is a transmembrane receptor expressed on platelets and several hematopoietic cells. CLEC2 regulates platelet aggregation and the immune response. We investigated its expression and function in normal and transformed gastric epithelial cells from human tissues. We performed tissue microarray analyses of gastric carcinoma samples collected from 96 patients who underwent surgery at Zhongshan Hospital of Fudan University in Shanghai, China and performed real-time polymerase chain reaction assays from an independent group of 60 patients; matched nontumor gastric mucosa tissues were used as the control. Full-length and mutant forms of CLEC2 were expressed in gastric cancer cell line (MGC80-3), or CLEC2 protein was knocked down using small-hairpin RNAs in gastric cancer cell lines (NCI-N87 and AGS). CLEC2 signaling was stimulated by incubation of cells with recombinant human podoplanin or an antibody agonist of CLEC2; cell migration and invasion were assessed by transwell and wound-healing assays. Immunoblot, immunofluorescence microscopy, and real-time polymerase chain reaction assays were used to measure expression of markers of the epithelial to mesenchymal transition and activation of signaling pathways. Immunoprecipitation experiments were performed with an antibody against spleen tyrosine kinase (SYK). Cells were injected into lateral tail vein of BALB/C nude mice; some mice were also given injections of the phosphoinositide 3-kinase (PI3K) inhibitor LY294002. Lung and liver tissues were collected and analyzed for metastases. Levels of CLEC2 were higher in nontumor gastric mucosa (control) than in gastric tumor samples. Levels of CLEC2 protein in gastric tumor tissues correlated with depth of tumor invasion, metastasis to lymph node, tumor TNM stage, and 5-year survival of patients. Activation of CLEC2 in gastric cancer cells reduced their invasive activities in vitro and expression of epithelial to mesenchymal transition

  10. Role of Nicotinic Acetylcholine Receptor α3 and α7 Subunits in Detrusor Overactivity Induced by Partial Bladder Outlet Obstruction in Rats

    Directory of Open Access Journals (Sweden)

    Hyo Sin Kim

    2015-03-01

    Full Text Available Purpose: To investigate the role of α3 and α7 nicotinic acetylcholine receptor subunits (nAChRs in the bladder, using a rat model with detrusor overactivity induced by partial bladder outlet obstruction (BOO. Methods: Forty Sprague-Dawley rats were used: 10 were sham-operated (control group and 30 were observed for 3 weeks after partial BOO. BOO-induced rats were further divided into 3 groups: Two groups of 10 rats each received intravesicular infusions with hexamethonium (HM group; n=10 or methyllycaconitine (MLC group; n=10, which are antagonists for α3 and α7 nAChRs, respectively. The remaining BOO-induced rats received only saline infusion (BOO group; n=10. Based on the contraction interval measurements using cystometrogram, the contraction pressure and nonvoiding bladder contractions were compared between the control and the three BOO-induced groups. Immunofluorescent staining and Western blotting were used to analyze α3 and α7 nAChRs levels. Results: The contraction interval of the MLC group was higher than that of the BOO group (P<0.05. Nonvoiding bladder contraction almost disappeared in the HM and MLC groups. Contraction pressure increased in the BOO group (P<0.05 compared with the control group and decreased in the HM and MLC groups compared with the BOO group (P<0.05. Immunofluorescence staining showed that the α3 nAChR signals increased in the urothelium, and the α7 nAChR signals increased in the urothelium and detrusor muscle of the BOO group compared with the control group. Western blot analysis showed that both α3 and α7 nAChR levels increased in the BOO group (P<0.05. Conclusions: Alpha3 and α7 nAChRs are associated with detrusor overactivity induced by BOO. Furthermore, nAChR antagonists could help in clinically improving detrusor overactivity.

  11. Induced expression of hepatic N-methyl-D-aspartate receptor 2C subunit gene during liver enlargement induced by lead nitrate, a hepatocellular mitogen.

    Science.gov (United States)

    Nemoto, Kiyomitsu; Ikeda, Ayaka; Hikida, Tokihiro; Kojima, Misaki; Degawa, Masakuni

    2013-02-01

    We previously demonstrated the super-induced expression of the Grin2c gene encoding the N-methyl-D-aspartate receptor 2C subunit during the development of liver enlargement with hepatocellular hypertrophy induced by phenobarbital, clofibrate, or piperonyl butoxide. In the present study, we assessed whether or not Grin2c gene expression was induced during the development of chemically induced liver enlargement with hyperplasia. Male Sprague-Dawley (SD) rats, stroke-prone spontaneously hypertensive rats (SHRSPs), and SHRSP's normotensive control, Wistar-Kyoto (WKY) rats, were administered lead nitrate (LN) (0.1 mmol/kg, single i.v.), a direct inducer of liver hyperplasia, and changes in the level of Grin2c mRNA in the liver were assessed by real-time RT-PCR. The level of hepatic Grin2c mRNA was significantly higher 6-48 hr after the injection in SD rats (about 30~40- and 70-fold over the control at 6~24 hr and 48 hr, respectively) and in WKY rats (about 20-fold over the control only at 12 hr), but was not significantly higher in SHRSPs. Such differences in LN-induced levels of Grin2c mRNA among SD rats, WKY rats, and SHRSPs were closely correlated with those in the previously reported increase in liver weight 48 hr after LN administration. The present findings suggest that the increase in the level of hepatic Grin2c mRNA relates to development of chemically induced liver enlargement with hyperplasia.

  12. Maternal Separation during Breastfeeding Induces Gender-Dependent Changes in Anxiety and the GABA-A Receptor Alpha-Subunit in Adult Wistar Rats.

    Directory of Open Access Journals (Sweden)

    Diego Armando León Rodríguez

    Full Text Available Different models of rodent maternal separation (MS have been used to investigate long-term neurobiological and behavioral changes, associated with early stress. However, few studies have involved the analysis of sex-related differences in central anxiety modulation. This study investigated whether MS during breastfeeding affected adult males and females in terms of anxiety and brain GABA-A receptor-alpha-subunit immunoreactivity. The brain areas analyzed were the amygdale (AM, hippocampus (HP, medial prefrontal cortex (mPFC, medial preoptic area (POA and paraventricular nucleus (PVN. Rats were housed under a reversed light/dark cycle (lights off at 7∶00 h with access to water and food ad libitum. Animals underwent MS twice daily during the dark cycle from postnatal day 1 to postnatal day 21. Behavior was tested when rats were 65-70 days old using the elevated plus maze and after brains were treated for immunohistochemistry. We found that separated females spent more time in the open arms and showed more head dipping behavior compared with controls. The separated males spent more time in the center of the maze and engaged in more stretching behavior than the controls. Immunohistochemistry showed that separated females had less immunostained cells in the HP, mPFC, PVN and POA, while separated males had fewer immunolabeled cells in the PFC, PVN and AM. These results could indicate that MS has gender-specific effects on anxiety behaviors and that these effects are likely related to developmental alterations involving GABA-A neurotransmission.

  13. Gestational chronodisruption impairs hippocampal expression of NMDA receptor subunits Grin1b/Grin3a and spatial memory in the adult offspring.

    Directory of Open Access Journals (Sweden)

    Nelson Vilches

    Full Text Available Epidemiological and experimental evidence correlates adverse intrauterine conditions with the onset of disease later in life. For a fetus to achieve a successful transition to extrauterine life, a myriad of temporally integrated humoral/biophysical signals must be accurately provided by the mother. We and others have shown the existence of daily rhythms in the fetus, with peripheral clocks being entrained by maternal cues, such as transplacental melatonin signaling. Among developing tissues, the fetal hippocampus is a key structure for learning and memory processing that may be anticipated as a sensitive target of gestational chronodisruption. Here, we used pregnant rats exposed to constant light treated with or without melatonin as a model of gestational chronodisruption, to investigate effects on the putative fetal hippocampus clock, as well as on adult offspring's rhythms, endocrine and spatial memory outcomes. The hippocampus of fetuses gestated under light:dark photoperiod (12:12 LD displayed daily oscillatory expression of the clock genes Bmal1 and Per2, clock-controlled genes Mtnr1b, Slc2a4, Nr3c1 and NMDA receptor subunits 1B-3A-3B. In contrast, in the hippocampus of fetuses gestated under constant light (LL, these oscillations were suppressed. In the adult LL offspring (reared in LD during postpartum, we observed complete lack of day/night differences in plasma melatonin and decreased day/night differences in plasma corticosterone. In the adult LL offspring, overall hippocampal day/night difference of gene expression was decreased, which was accompanied by a significant deficit of spatial memory. Notably, maternal melatonin replacement to dams subjected to gestational chronodisruption prevented the effects observed in both, LL fetuses and adult LL offspring. Collectively, the present data point to adverse effects of gestational chronodisruption on long-term cognitive function; raising challenging questions about the consequences of

  14. Kir6.2 activation by sulfonylurea receptors: a different mechanism of action for SUR1 and SUR2A subunits via the same residues

    Science.gov (United States)

    Principalli, Maria A; Dupuis, Julien P; Moreau, Christophe J; Vivaudou, Michel; Revilloud, Jean

    2015-01-01

    ATP-sensitive potassium channels (K-ATP channels) play a key role in adjusting the membrane potential to the metabolic state of cells. They result from the unique combination of two proteins: the sulfonylurea receptor (SUR), an ATP-binding cassette (ABC) protein, and the inward rectifier K+ channel Kir6.2. Both subunits associate to form a heterooctamer (4 SUR/4 Kir6.2). SUR modulates channel gating in response to the binding of nucleotides or drugs and Kir6.2 conducts potassium ions. The activity of K-ATP channels varies with their localization. In pancreatic β-cells, SUR1/Kir6.2 channels are partly active at rest while in cardiomyocytes SUR2A/Kir6.2 channels are mostly closed. This divergence of function could be related to differences in the interaction of SUR1 and SUR2A with Kir6.2. Three residues (E1305, I1310, L1313) located in the linker region between transmembrane domain 2 and nucleotide-binding domain 2 of SUR2A were previously found to be involved in the activation pathway linking binding of openers onto SUR2A and channel opening. To determine the role of the equivalent residues in the SUR1 isoform, we designed chimeras between SUR1 and the ABC transporter multidrug resistance-associated protein 1 (MRP1), and used patch clamp recordings on Xenopus oocytes to assess the functionality of SUR1/MRP1 chimeric K-ATP channels. Our results reveal that the same residues in SUR1 and SUR2A are involved in the functional association with Kir6.2, but they display unexpected side-chain specificities which could account for the contrasted properties of pancreatic and cardiac K-ATP channels. PMID:26416970

  15. Contribution of NMDA receptor hypofunction in prefrontal and cortical excitatory neurons to schizophrenia-like phenotypes.

    Directory of Open Access Journals (Sweden)

    Gregory R Rompala

    Full Text Available Pharmacological and genetic studies support a role for NMDA receptor (NMDAR hypofunction in the etiology of schizophrenia. We have previously demonstrated that NMDAR obligatory subunit 1 (GluN1 deletion in corticolimbic interneurons during early postnatal development is sufficient to confer schizophrenia-like phenotypes in mice. However, the consequence of NMDAR hypofunction in cortical excitatory neurons is not well delineated. Here, we characterize a conditional knockout mouse strain (CtxGluN1 KO mice, in which postnatal GluN1 deletion is largely confined to the excitatory neurons in layer II/III of the medial prefrontal cortex and sensory cortices, as evidenced by the lack of GluN1 mRNA expression in in situ hybridization immunocytochemistry as well as the lack of NMDA currents with in vitro recordings. Mutants were impaired in prepulse inhibition of the auditory startle reflex as well as object-based short-term memory. However, they did not exhibit impairments in additional hallmarks of schizophrenia-like phenotypes (e.g. spatial working memory, social behavior, saccharine preference, novelty and amphetamine-induced hyperlocomotion, and anxiety-related behavior. Furthermore, upon administration of the NMDA receptor antagonist, MK-801, there were no differences in locomotor activity versus controls. The mutant mice also showed negligible levels of reactive oxygen species production following chronic social isolation, and recording of miniature-EPSC/IPSCs from layer II/III excitatory neurons in medial prefrontal cortex suggested no alteration in GABAergic activity. All together, the mutant mice displayed cognitive deficits in the absence of additional behavioral or cellular phenotypes reflecting schizophrenia pathophysiology. Thus, NMDAR hypofunction in prefrontal and cortical excitatory neurons may recapitulate only a cognitive aspect of human schizophrenia symptoms.

  16. Contribution of NMDA receptor hypofunction in prefrontal and cortical excitatory neurons to schizophrenia-like phenotypes.

    Science.gov (United States)

    Rompala, Gregory R; Zsiros, Veronika; Zhang, Shuqin; Kolata, Stefan M; Nakazawa, Kazu

    2013-01-01

    Pharmacological and genetic studies support a role for NMDA receptor (NMDAR) hypofunction in the etiology of schizophrenia. We have previously demonstrated that NMDAR obligatory subunit 1 (GluN1) deletion in corticolimbic interneurons during early postnatal development is sufficient to confer schizophrenia-like phenotypes in mice. However, the consequence of NMDAR hypofunction in cortical excitatory neurons is not well delineated. Here, we characterize a conditional knockout mouse strain (CtxGluN1 KO mice), in which postnatal GluN1 deletion is largely confined to the excitatory neurons in layer II/III of the medial prefrontal cortex and sensory cortices, as evidenced by the lack of GluN1 mRNA expression in in situ hybridization immunocytochemistry as well as the lack of NMDA currents with in vitro recordings. Mutants were impaired in prepulse inhibition of the auditory startle reflex as well as object-based short-term memory. However, they did not exhibit impairments in additional hallmarks of schizophrenia-like phenotypes (e.g. spatial working memory, social behavior, saccharine preference, novelty and amphetamine-induced hyperlocomotion, and anxiety-related behavior). Furthermore, upon administration of the NMDA receptor antagonist, MK-801, there were no differences in locomotor activity versus controls. The mutant mice also showed negligible levels of reactive oxygen species production following chronic social isolation, and recording of miniature-EPSC/IPSCs from layer II/III excitatory neurons in medial prefrontal cortex suggested no alteration in GABAergic activity. All together, the mutant mice displayed cognitive deficits in the absence of additional behavioral or cellular phenotypes reflecting schizophrenia pathophysiology. Thus, NMDAR hypofunction in prefrontal and cortical excitatory neurons may recapitulate only a cognitive aspect of human schizophrenia symptoms.

  17. Structural determinants of alpha-bungarotoxin binding to the sequence segment 181-200 of the muscle nicotinic acetylcholine receptor α subunit: Effects of cysteine/cystine modification and species-specific amino acid substitution

    International Nuclear Information System (INIS)

    McLane, K.E.; Wu, Xiadong; Diethelm, B.; Conti-Tronconi, B.M.

    1991-01-01

    The sequence segment 181-200 of the Torpedo nicotinic acetylcholine receptor (nAChR) αsubunit forms a binding site for α-bungarotoxin (α-BTX). Synthetic peptides corresponding to the homologous sequences of human, calf, mouse, chicken, frog, and cobra muscle nAChR α1 subunits were tested for their ability to bind 125 I-α-BTX, and differences in α-BTX affinity were determined by using solution (IC 50 s) and solid-phase (K d s) assays. Panels of overlapping peptides corresponding to the complete α1 subunit of mouse and human were also tested for α-BTX binding, but other sequence segments forming the α-BTX site were not consistently detectable. The role of a putative vicinal disulfide bound between Cys-192 and -193, relative to the Torpedo sequence, was determined by modifying the peptides with sulfhydryl reagents. Reduction and alkylation of the peptides decreased α-BTX binding, whereas oxidation of the peptides had little effect. These results indicate that while the adjacent cysteines are likely to be involved in forming the toxin/α1-subunit interface a vicinal disulfide bound was not required for α-BTX binding

  18. Connexins and M3 Muscarinic Receptors Contribute to Heterogeneous Ca2+ Signaling in Mouse Aortic Endothelium

    Directory of Open Access Journals (Sweden)

    François-Xavier Boittin

    2013-02-01

    Full Text Available Background/Aims: Smooth muscle tone is controlled by Ca2+ signaling in the endothelial layer. Mouse endothelial cells are interconnected by gap junctions made of Connexin40 (Cx40 and Cx37, which allow the exchange of signaling molecules to coordinate their activity. Here, we investigated the role of Cx40 in the endothelial Ca2+ signaling of the mouse aorta. Methods: Ca2+ imaging was performed on intact aortic endothelium from both wild type (Cx40+/+ and Connexin40-deficient (Cx40 -/- mice. Results: Acetylcholine (ACh induced early fast and high amplitude Ca2+ transients in a fraction of endothelial cells expressing the M3 muscarinic receptors. Inhibition of intercellular communication using carbenoxolone or octanol fully blocked the propagation of ACh-induced Ca2+ transients toward adjacent cells in WT and Cx40-/- mice. As compared to WT, Cx40-/- mice displayed a reduced propagation of ACh-induced Ca2+ waves, indicating that Cx40 contributes to the spreading of Ca2+ signals. The propagation of those Ca2+ responses was not blocked by suramin, a blocker of purinergic ATP receptors, indicating that there is no paracrine effect of ATP release on the Ca2+ waves. Conclusions: Altogether our data show that Cx40 and Cx37 contribute to the propagation and amplification of the Ca2+ signaling triggered by ACh in endothelial cells expressing the M3 muscarinic receptors.

  19. Contributions of sex, testosterone, and androgen receptor CAG repeat number to virtual Morris water maze performance.

    Science.gov (United States)

    Nowak, Nicole T; Diamond, Michael P; Land, Susan J; Moffat, Scott D

    2014-03-01

    The possibility that androgens contribute to the male advantage typically found on measures of spatial cognition has been investigated using a variety of approaches. To date, evidence to support the notion that androgens affect spatial cognition in healthy young adults is somewhat equivocal. The present study sought to clarify the association between testosterone (T) and spatial performance by extending measurements of androgenicity to include both measures of circulating T as well as an androgen receptor-specific genetic marker. The aims of this study were to assess the contributions of sex, T, and androgen receptor CAG repeat number (CAGr) on virtual Morris water task (vMWT) performance in a group of healthy young men and women. The hypothesis that men would outperform women on vMWT outcomes was supported. Results indicate that CAGr may interact with T to impact navigation performance and suggest that consideration of androgen receptor sensitivity is an important consideration in evaluating hormone-behavior relationships. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. Systematic study of association of four GABAergic genes: glutamic acid decarboxylase 1 gene, glutamic acid decarboxylase 2 gene, GABA(B) receptor 1 gene and GABA(A) receptor subunit beta2 gene, with schizophrenia using a universal DNA microarray.

    Science.gov (United States)

    Zhao, Xu; Qin, Shengying; Shi, Yongyong; Zhang, Aiping; Zhang, Jing; Bian, Li; Wan, Chunling; Feng, Guoyin; Gu, Niufan; Zhang, Guangqi; He, Guang; He, Lin

    2007-07-01

    Several studies have suggested the dysfunction of the GABAergic system as a risk factor in the pathogenesis of schizophrenia. In the present study, case-control association analysis was conducted in four GABAergic genes: two glutamic acid decarboxylase genes (GAD1 and GAD2), a GABA(A) receptor subunit beta2 gene (GABRB2) and a GABA(B) receptor 1 gene (GABBR1). Using a universal DNA microarray procedure we genotyped a total of 20 SNPs on the above four genes in a study involving 292 patients and 286 controls of Chinese descent. Statistically significant differences were observed in the allelic frequencies of the rs187269C/T polymorphism in the GABRB2 gene (P=0.0450, chi(2)=12.40, OR=1.65) and the -292A/C polymorphism in the GAD1 gene (P=0.0450, chi(2)=14.64 OR=1.77). In addition, using an electrophoretic mobility shift assay (EMSA), we discovered differences in the U251 nuclear protein binding to oligonucleotides representing the -292 SNP on the GAD1 gene, which suggests that the -292C allele has reduced transcription factor binding efficiency compared with the 292A allele. Using the multifactor-dimensionality reduction method (MDR), we found that the interactions among the rs187269C/T polymorphism in the GABRB2 gene, the -243A/G polymorphism in the GAD2 gene and the 27379C/T and 661C/T polymorphisms in the GAD1 gene revealed a significant association with schizophrenia (Pschizophrenia in the Chinese population.

  1. GluN2C/GluN2D subunit-selective NMDA receptor potentiator CIQ reverses MK-801-induced impairment in prepulse inhibition and working memory in Y-maze test in mice

    Science.gov (United States)

    Suryavanshi, P S; Ugale, R R; Yilmazer-Hanke, D; Stairs, D J; Dravid, S M

    2014-01-01

    Background and Purpose Despite ample evidence supporting the N-methyl-d-aspartate receptor (NMDAR) hypofunction hypothesis of schizophrenia, progress in the development of effective therapeutics based on this hypothesis has been limited. Facilitation of NMDA receptor function by co-agonists (d-serine or glycine) only partially alleviates the symptoms in schizophrenia; other means to facilitate NMDA receptors are required. NMDA receptor sub-types differ in their subunit composition, with varied GluN2 subunits (GluN2A-GluN2D) imparting different physiological, biochemical and pharmacological properties. CIQ is a positive allosteric modulator that is selective for GluN2C/GluN2D-containing NMDA receptors (Mullasseril et al.). Experimental Approach The effect of systemic administration of CIQ was tested on impairment in prepulse inhibition (PPI), hyperlocomotion and stereotypy induced by i.p. administration of MK-801 and methamphetamine. The effect of CIQ was also tested on MK-801-induced impairment in working memory in Y-maze spontaneous alternation test. Key Results We found that systemic administration of CIQ (20 mg·kg−1, i.p.) in mice reversed MK-801 (0.15 mg·kg−1, i.p.)-induced, but not methamphetamine (3 mg·kg−1, i.p.)-induced, deficit in PPI. MK-801 increased the startle amplitude to pulse alone, which was not reversed by CIQ. In contrast, methamphetamine reduced the startle amplitude to pulse alone, which was reversed by CIQ. CIQ also partially attenuated MK-801- and methamphetamine-induced hyperlocomotion and stereotyped behaviours. Additionally, CIQ reversed the MK-801-induced working memory deficit in spontaneous alternation in a Y-maze. Conclusion and Implications Together, these results suggest that facilitation of GluN2C/GluN2D-containing receptors may serve as an important therapeutic strategy for treating positive and cognitive symptoms in schizophrenia. PMID:24236947

  2. Lessons from crystal structures of kainate receptors

    DEFF Research Database (Denmark)

    Møllerud, Stine; Frydenvang, Karla Andrea; Pickering, Darryl S

    2017-01-01

    Kainate receptors belong to the family of ionotropic glutamate receptors. These receptors assemble from five subunits (GluK1-5) into tetrameric ion channels. Kainate receptors are located at both pre- and postsynaptic membranes in the central nervous system where they contribute to excitatory...... synaptic transmission and modulate network excitability by regulating neurotransmitter release. Dysfunction of kainate receptors has been implicated in several neurological disorders such as epilepsy, schizophrenia and depression. Here we provide a review on the current understanding of kainate receptor...

  3. Effects of cigarette smoke exposure on nicotinic acetylcholine receptor subunits {alpha}7 and {beta}2 in the sudden infant death syndrome (SIDS) brainstem

    Energy Technology Data Exchange (ETDEWEB)

    Machaalani, Rita, E-mail: rita.machaalani@sydney.edu.au [Department of Medicine, The University of Sydney, NSW 2006 (Australia); Bosch Institute, The University of Sydney, NSW 2006 (Australia); The Children' s Hospital at Westmead, NSW 2145 (Australia); Say, Meichien [Department of Medicine, The University of Sydney, NSW 2006 (Australia); Bosch Institute, The University of Sydney, NSW 2006 (Australia); Waters, Karen A. [Department of Medicine, The University of Sydney, NSW 2006 (Australia); Bosch Institute, The University of Sydney, NSW 2006 (Australia); The Children' s Hospital at Westmead, NSW 2145 (Australia)

    2011-12-15

    It is postulated that nicotine, as the main neurotoxic constituent of cigarette smoke, influences SIDS risk through effects on nicotinic acetylcholine receptors (nAChRs) in brainstem nuclei that control respiration and arousal. This study compared {alpha}7 and {beta}2 nAChR subunit expression in eight nuclei of the caudal and rostral medulla and seven nuclei of the pons between SIDS (n = 46) and non-SIDS infants (n = 14). Evaluation for associations with known SIDS risk factors included comparison according to whether infants had a history of exposure to cigarette smoke in the home, and stratification for sleep position and gender. Compared to non-SIDS infants, SIDS infants had significantly decreased {alpha}7 in the caudal nucleus of the solitary tract (cNTS), gracile and cuneate nuclei, with decreased {beta}2 in the cNTS and increased {beta}2 in the facial. When considering only the SIDS cohort: 1-cigarette smoke exposure was associated with increased {alpha}7 in the vestibular nucleus and increased {beta}2 in the rostral dorsal motor nucleus of the vagus, rNTS and Cuneate, 2-there was a gender interaction for {alpha}7 in the gracile and cuneate, and {beta}2 in the cNTS and rostral arcuate nucleus, and 3-there was no effect of sleep position on {alpha}7, but prone sleep was associated with decreased {beta}2 in three nuclei of the pons. In conclusion, SIDS infants demonstrate differences in expression of {alpha}7 and {beta}2 nAChRs within brainstem nuclei that control respiration and arousal, which is independent on prior history of cigarette smoke exposure, especially for the NTS, with additional differences for smoke exposure ({beta}2), gender ({alpha}7 and {beta}2) and sleep position ({beta}2) evident. -- Highlights: Black-Right-Pointing-Pointer The 'normal' response to smoke exposure is decreased {alpha}7 and {beta}2 in certain nuclei. Black-Right-Pointing-Pointer SIDS infants have decreased {alpha}7 in cNTS, Grac and Cun. Black

  4. Effects of cigarette smoke exposure on nicotinic acetylcholine receptor subunits α7 and β2 in the sudden infant death syndrome (SIDS) brainstem

    International Nuclear Information System (INIS)

    Machaalani, Rita; Say, Meichien; Waters, Karen A.

    2011-01-01

    It is postulated that nicotine, as the main neurotoxic constituent of cigarette smoke, influences SIDS risk through effects on nicotinic acetylcholine receptors (nAChRs) in brainstem nuclei that control respiration and arousal. This study compared α7 and β2 nAChR subunit expression in eight nuclei of the caudal and rostral medulla and seven nuclei of the pons between SIDS (n = 46) and non-SIDS infants (n = 14). Evaluation for associations with known SIDS risk factors included comparison according to whether infants had a history of exposure to cigarette smoke in the home, and stratification for sleep position and gender. Compared to non-SIDS infants, SIDS infants had significantly decreased α7 in the caudal nucleus of the solitary tract (cNTS), gracile and cuneate nuclei, with decreased β2 in the cNTS and increased β2 in the facial. When considering only the SIDS cohort: 1—cigarette smoke exposure was associated with increased α7 in the vestibular nucleus and increased β2 in the rostral dorsal motor nucleus of the vagus, rNTS and Cuneate, 2—there was a gender interaction for α7 in the gracile and cuneate, and β2 in the cNTS and rostral arcuate nucleus, and 3—there was no effect of sleep position on α7, but prone sleep was associated with decreased β2 in three nuclei of the pons. In conclusion, SIDS infants demonstrate differences in expression of α7 and β2 nAChRs within brainstem nuclei that control respiration and arousal, which is independent on prior history of cigarette smoke exposure, especially for the NTS, with additional differences for smoke exposure (β2), gender (α7 and β2) and sleep position (β2) evident. -- Highlights: ► The ‘normal’ response to smoke exposure is decreased α7 and β2 in certain nuclei. ► SIDS infants have decreased α7 in cNTS, Grac and Cun. ► SIDS infants have decreased β2 in cNTS and increased β2 in facial. ► The NTS is more sensitive to both α7 and β2 regulation in SIDS. ► Smoke exposure

  5. Adaptor protein 1 B mu subunit does not contribute to the recycling of kAE1 protein in polarized renal epithelial cells.

    Science.gov (United States)

    Almomani, Ensaf Y; Touret, Nicolas; Cordat, Emmanuelle

    2018-04-13

    Mutations in the gene encoding the kidney anion exchanger 1 (kAE1) can lead to distal renal tubular acidosis (dRTA). dRTA mutations reported within the carboxyl (C)-terminal tail of kAE1 result in apical mis-targeting of the exchanger in polarized renal epithelial cells. As kAE1 physically interacts with the μ subunit of epithelial adaptor protein 1 B (AP-1B), we investigated the role of heterologously expressed μ1B subunit of the AP-1B complex for kAE1 retention to the basolateral membrane in polarized porcine LLC-PK1 renal epithelial cells that are devoid of endogenous AP-1B. We confirmed the interaction and close proximity between kAE1 and μ1B using immunoprecipitation and proximity ligation assay, respectively. Expressing the human μ1B subunit in these cells decreased significantly the amount of cell surface kAE1 at the steady state, but had no significant effect on kAE1 recycling and endocytosis. We show that (i) heterologous expression of μ1B displaces the physical interaction of endogenous GAPDH with kAE1 WT supporting that both AP-1B and GAPDH proteins bind to an overlapping site on kAE1 and (ii) phosphorylation of tyrosine 904 within the potential YDEV interaction motif does not alter the kAE1/AP-1B interaction. We conclude that μ1B subunit is not involved in recycling of kAE1.

  6. The vertebrate ancestral repertoire of visual opsins, transducin alpha subunits and oxytocin/vasopressin receptors was established by duplication of their shared genomic region in the two rounds of early vertebrate genome duplications.

    Science.gov (United States)

    Lagman, David; Ocampo Daza, Daniel; Widmark, Jenny; Abalo, Xesús M; Sundström, Görel; Larhammar, Dan

    2013-11-02

    Vertebrate color vision is dependent on four major color opsin subtypes: RH2 (green opsin), SWS1 (ultraviolet opsin), SWS2 (blue opsin), and LWS (red opsin). Together with the dim-light receptor rhodopsin (RH1), these form the family of vertebrate visual opsins. Vertebrate genomes contain many multi-membered gene families that can largely be explained by the two rounds of whole genome duplication (WGD) in the vertebrate ancestor (2R) followed by a third round in the teleost ancestor (3R). Related chromosome regions resulting from WGD or block duplications are said to form a paralogon. We describe here a paralogon containing the genes for visual opsins, the G-protein alpha subunit families for transducin (GNAT) and adenylyl cyclase inhibition (GNAI), the oxytocin and vasopressin receptors (OT/VP-R), and the L-type voltage-gated calcium channels (CACNA1-L). Sequence-based phylogenies and analyses of conserved synteny show that the above-mentioned gene families, and many neighboring gene families, expanded in the early vertebrate WGDs. This allows us to deduce the following evolutionary scenario: The vertebrate ancestor had a chromosome containing the genes for two visual opsins, one GNAT, one GNAI, two OT/VP-Rs and one CACNA1-L gene. This chromosome was quadrupled in 2R. Subsequent gene losses resulted in a set of five visual opsin genes, three GNAT and GNAI genes, six OT/VP-R genes and four CACNA1-L genes. These regions were duplicated again in 3R resulting in additional teleost genes for some of the families. Major chromosomal rearrangements have taken place in the teleost genomes. By comparison with the corresponding chromosomal regions in the spotted gar, which diverged prior to 3R, we could time these rearrangements to post-3R. We present an extensive analysis of the paralogon housing the visual opsin, GNAT and GNAI, OT/VP-R, and CACNA1-L gene families. The combined data imply that the early vertebrate WGD events contributed to the evolution of vision and the

  7. Contributions of conserved residues at the gating interface of glycine receptors

    DEFF Research Database (Denmark)

    Pless, Stephan Alexander; Leung, Ada W Y; Galpin, Jason D

    2011-01-01

    and the in vivo nonsense suppression method to incorporate unnatural amino acids to probe the electrostatic and hydrophobic contributions of five highly conserved side chains near the interface, Glu-53, Phe-145, Asp-148, Phe-187, and Arg-218. Our results suggest a salt bridge between Asp-148 in loop 7 and Arg-218......Glycine receptors (GlyRs) are chloride channels that mediate fast inhibitory neurotransmission and are members of the pentameric ligand-gated ion channel (pLGIC) family. The interface between the ligand binding domain and the transmembrane domain of pLGICs has been proposed to be crucial...

  8. Glutamatergic receptor dysfunction in spinal cord contributes to the exaggerated exercise pressor reflex in heart failure.

    Science.gov (United States)

    Wang, Han-Jun; Cahoon, Rebecca; Cahoon, Edgar B; Zheng, Hong; Patel, Kaushik P; Zucker, Irving H

    2015-03-01

    Excitatory amino acids (e.g., glutamate) released by contraction-activated skeletal muscle afferents into the dorsal horn of the spinal cord initiate the central component of the exercise pressor reflex (EPR) in physiological conditions. However, the role of glutamate and glutamate receptors in mediating the exaggerated EPR in the chronic heart failure (CHF) state remains to be determined. In the present study, we performed microinjection of glutamate receptor antagonists into ipisilateral L4/L5 dorsal horns to investigate their effects on the pressor response to static contraction induced by stimulation of the peripheral end of L4/L5 ventral roots in decerebrate sham-operated (sham) and CHF rats. Microinjection of glutamate (10 mM, 100 nl) into the L4 or L5 dorsal horn caused a greater pressor response in CHF rats compared with sham rats. Furthermore, microinjection of either the broad-spectrum glutamate receptor antagonist kynurenate (10 mM, 100 nl) or the N-methyl-d-aspartate (NMDA) receptor antagonist dl-2-amino-5-phosphonovalerate (50 mM, 100 nl) or the non-NMDA-sensitive receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (5 mM, 100 nl) into L4/5 dorsal horns decreased the pressor response to static contraction in CHF rats to a greater extent than in sham rats. Molecular evidence showed that the protein expression of glutamate receptors (both non-NMDA and NMDA) was elevated in the dorsal horn of the lumbar spinal cord in CHF rats. In addition, data from microdialysis experiments demonstrated that although basal glutamate release at the dorsal horn at rest was similar between sham and CHF rats (225 ± 50 vs. 260 ± 63 nM in sham vs. CHF rats, n = 4, P > 0.05), CHF rats exhibit greater glutamate release into the dorsal horn during muscle contraction compared with sham rats (549 ± 60 vs. 980 ± 65 nM in sham vs. CHF rats, n = 4, P < 0.01). These data indicate that the spinal glutamate system contributes to the exaggerated EPR in the CHF state. Copyright

  9. A dopamine receptor contributes to paraquat-induced neurotoxicity in Drosophila

    Science.gov (United States)

    Cassar, Marlène; Issa, Abdul-Raouf; Riemensperger, Thomas; Petitgas, Céline; Rival, Thomas; Coulom, Hélène; Iché-Torres, Magali; Han, Kyung-An; Birman, Serge

    2015-01-01

    Long-term exposure to environmental oxidative stressors, like the herbicide paraquat (PQ), has been linked to the development of Parkinson's disease (PD), the most frequent neurodegenerative movement disorder. Paraquat is thus frequently used in the fruit fly Drosophila melanogaster and other animal models to study PD and the degeneration of dopaminergic neurons (DNs) that characterizes this disease. Here, we show that a D1-like dopamine (DA) receptor, DAMB, actively contributes to the fast central nervous system (CNS) failure induced by PQ in the fly. First, we found that a long-term increase in neuronal DA synthesis reduced DAMB expression and protected against PQ neurotoxicity. Secondly, a striking age-related decrease in PQ resistance in young adult flies correlated with an augmentation of DAMB expression. This aging-associated increase in oxidative stress vulnerability was not observed in a DAMB-deficient mutant. Thirdly, targeted inactivation of this receptor in glutamatergic neurons (GNs) markedly enhanced the survival of Drosophila exposed to either PQ or neurotoxic levels of DA, whereas, conversely, DAMB overexpression in these cells made the flies more vulnerable to both compounds. Fourthly, a mutation in the Drosophila ryanodine receptor (RyR), which inhibits activity-induced increase in cytosolic Ca2+, also strongly enhanced PQ resistance. Finally, we found that DAMB overexpression in specific neuronal populations arrested development of the fly and that in vivo stimulation of either DNs or GNs increased PQ susceptibility. This suggests a model for DA receptor-mediated potentiation of PQ-induced neurotoxicity. Further studies of DAMB signaling in Drosophila could have implications for better understanding DA-related neurodegenerative disorders in humans. PMID:25158689

  10. The LIM domain protein FHL2 interacts with the NR5A family of nuclear receptors and CREB to activate the inhibin-α subunit gene in ovarian granulosa cells.

    Science.gov (United States)

    Matulis, Christina K; Mayo, Kelly E

    2012-08-01

    Nuclear receptor transcriptional activity is enhanced by interaction with coactivators. The highly related nuclear receptor 5A (NR5A) subfamily members liver receptor homolog 1 and steroidogenic factor 1 bind to and activate several of the same genes, many of which are important for reproductive function. To better understand transcriptional activation by these nuclear receptors, we sought to identify interacting proteins that might function as coactivators. The LIM domain protein four and a half LIM domain 2 (FHL2) was identified as interacting with the NR5A receptors in a yeast two-hybrid screen of a human ovary cDNA library. FHL2, and the closely related FHL1, are both expressed in the rodent ovary and in granulosa cells. Small interfering RNA-mediated knockdown of FHL1 and FHL2 in primary mouse granulosa cells reduced expression of the NR5A target genes encoding inhibin-α and P450scc. In vitro assays confirmed the interaction between the FHL and NR5A proteins and revealed that a single LIM domain of FHL2 is sufficient for this interaction, whereas determinants in both the ligand binding domain and DNA binding domain of NR5A proteins are important. FHL2 enhances the ability of both liver receptor homolog 1 and steroidogenic factor 1 to activate the inhibin-α subunit gene promoter in granulosa cells and thus functions as a transcriptional coactivator. FHL2 also interacts with cAMP response element-binding protein and substantially augments activation of inhibin gene expression by the combination of NR5A receptors and forskolin, suggesting that FHL2 may facilitate integration of these two signals. Collectively these results identify FHL2 as a novel coactivator of NR5A nuclear receptors in ovarian granulosa cells and suggest its involvement in regulating target genes important for mammalian reproduction.

  11. Acute Inflammation Increases Pituitary and Hypothalamic Glycoprotein Hormone Subunit B5 mRNA Expression in Association with Decreased Thyrotrophin Receptor mRNA Expression in Mice

    NARCIS (Netherlands)

    van Zeijl, C. J. J.; Surovtseva, O. V.; Wiersinga, W. M.; Fliers, E.; Boelen, A.

    2011-01-01

    The biological function of thyrostimulin, consisting of the GPA2 and GPB5 subunit, is currently poorly understood. The recent observation that pro-inflammatory cytokines up-regulate the transcription of GPB5 in vitro suggested a role for thyrostimulin in the nonthyroidal illness syndrome, a state of

  12. Glutamate receptor antibodies directed against AMPA receptors subunit 3 peptide B (GluR3B) can be produced in DBA/2J mice, lower seizure threshold and induce abnormal behavior.

    Science.gov (United States)

    Ganor, Yonatan; Goldberg-Stern, Hadassa; Cohen, Ran; Teichberg, Vivian; Levite, Mia

    2014-04-01

    Anti-GluR3B antibodies (GluR3B Ab's), directed against peptide B/aa372-395 of GluR3 subunit of glutamate/AMPA receptors, are found in ∼35% of epilepsy patients, activate glutamate/AMPA receptors, evoke ion currents, kill neurons and damage the brain. We recently found that GluR3B Ab's also associate with neurological/psychiatric/behavioral abnormalities in epilepsy patients. Here we asked if GluR3B Ab's could be produced in DBA/2J mice, and also modulate seizure threshold and/or cause behavioral/motor impairments in these mice. DBA/2J mice were immunized with the GluR3B peptide in Complete Freund's Adjuvant (CFA), or with controls: ovalbumin (OVA), CFA, or phosphate-buffer saline (PBS). GluR3B Ab's and OVA Ab's were tested. Seizures were induced in all mice by the chemoconvulsant pentylenetetrazole (PTZ) at three time points, each time with less PTZ to avoid non-specific death. Behavior was examined in Open-Field, RotaRod and Grip tests. GluR3B Ab's were produced only in GluR3B-immunized mice, while OVA Ab's were produced only in OVA-immunized mice, showing high Ab's specificity. In GluR3B Ab's negative mice, seizure severity scores and percentages of animals developing generalized seizures declined in response to decreasing PTZ doses. In contrast, both parameters remained unchanged/high in the GluR3B Ab's positive mice, showing that these mice were more susceptible to seizures. The seizure scores associated significantly with the GluR3B Ab's levels. GluR3B Ab's positive mice were also more anxious in Open-Field test, fell faster in RotaRod test, and fell more in Grip test, compared to all the control mice. GluR3B Ab's are produced in DBA/2J mice, facilitate seizures and induce behavioral/motor impairments. This animal model can therefore serve for studying autoimmune epilepsy and abnormal behavior mediated by pathogenic anti-GluR3B Ab's. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Synthesis and preclinical evaluation of carbon-11 labelled N-((5-(4-fluoro-2-[11C]methoxyphenyl)pyridin-3-yl)methyl)cyclopentanamine as a PET tracer for NR2B subunit-containing NMDA receptors

    International Nuclear Information System (INIS)

    Christiaans, Johannes A.M.; Klein, Pieter J.; Metaxas, Athanasios; Kooijman, Esther J.M.; Schuit, Robert C.; Leysen, Josée E.; Lammertsma, Adriaan A.; Berckel, Bart N.M. van; Windhorst, Albert D.

    2014-01-01

    Introduction: The N-methyl-D-Aspartate (NMDA) receptor plays an important role in learning and memory. Overactivation is thought to play an important role in neurodegenerative disorders such as Alzheimer's disease. Currently, it is not possible to assess N-methyl-D-aspartate receptor (NMDAr) bio-availability in vivo. The purpose of this study was to develop a positron emission tomography (PET) ligand for the NR2B binding site of the NMDA receptor. Methods: N-((5-(4-fluoro-2-methoxyphenyl)pyridin-3-yl)methyl)cyclopentanamine was radiolabelled with carbon-11 in the phenyl moiety. Biodistribution and blocking studies were carried out in anaesthetized mice and in non-anaesthetized rats. Results: N-((5-(4-fluoro-2-[ 11 C]methoxyphenyl)pyridin-3-yl)methyl)cyclopentanamine was prepared in 49 ± 3% (decay-corrected) yield, affording 4.1 ± 0.3 GBq of formulated product at the end of synthesis with a radiochemical purity of > 99% and with a specific activity of 78 ± 10 GBq/μmol. Conclusion: A new NR2B PET ligand was developed in high yield. [ 11 C]4 readily enters the brain and binds to the NR2B subunit-containing NMDAr in the rodent brain. High sigma-1 receptor binding may, however, limit its future application as a PET probe for imaging the NR2B subunit-containing NMDAr. Anaesthesia has an effect on NMDAr function and therefore can complicate interpretation of preclinical in vivo results. In addition, effects of endogenous compounds cannot be excluded. Despite these potential limitations, further studies are warranted to investigate the values of [ 11 C]4 as an NR2B PET ligand

  14. Influence of early life status epilepticus on the developmental expression profile of the GluA2 subunit of AMPA receptors

    Czech Academy of Sciences Publication Activity Database

    Szczurowska, Ewa; Ergang, Peter; Kubová, Hana; Druga, Rastislav; Salaj, M.; Mareš, Pavel

    2016-01-01

    Roč. 283, Part A (2016), s. 97-109 ISSN 0014-4886 R&D Projects: GA ČR(CZ) GA15-16605S; GA ČR(CZ) GBP304/12/G069 Institutional support: RVO:67985823 Keywords : development * pilocarpine * status epilepticus * LiCl * AMPA * GluA2 * subunit * expression * GRIA2A Subject RIV: FH - Neurology Impact factor: 4.706, year: 2016

  15. α/sub i/-3 cDNA encodes the α subunit of G/sub k/, the stimulatory G protein of receptor-regulated K+ channels

    International Nuclear Information System (INIS)

    Codina, J.; Olate, J.; Abramowitz, J.; Mattera, R.; Cook, R.G.; Birnbaumer, L.

    1988-01-01

    cDNA cloning has identified the presence in the human genome of three genes encoding α subunits of pertussis toxin substrates, generically called G/sub i/. They are named α/sub i/-1, α/sub i/-2 and α/sub i/-3. However, none of these genes has been functionally identified with any of the α subunits of several possible G proteins, including pertussis toxin-sensitive G/sub p/'s, stimulatory to phospholipase C or A 2 , G/sub i/, inhibitory to adenylyl cyclase, or G/sub k/, stimulatory to a type of K + channels. The authors now report the nucleotide sequence and the complete predicted amino acid sequence of human liver α/sub i/-3 and the partial amino acid sequence of proteolytic fragments of the α subunit of human erythrocyte G/sub k/. The amino acid sequence of the proteolytic fragment is uniquely encoded by the cDNA of α/sub i/-3, thus identifying it as α/sub k/. The probable identity of α/sub i/-1 with α/sub p/ and possible roles for α/sub i/-2, as well as additional roles for α/sub i/-1 and α/sub i/-3 (α/sub k/) are discussed

  16. Bioassays for TSH Receptor Autoantibodies, from FRTL-5 Cells to TSH Receptor-LH/CG Receptor Chimeras: The Contribution of Leonard D. Kohn.

    Science.gov (United States)

    Giuliani, Cesidio; Saji, Motoyasu; Bucci, Ines; Napolitano, Giorgio

    2016-01-01

    Since the discovery 60 years ago of the "long-acting thyroid stimulator" by Adams and Purves, great progress has been made in the detection of thyroid-stimulating hormone (TSH) receptor (TSHR) autoantibodies (TRAbs) in Graves' disease. Today, commercial assays are available that can detect TRAbs with high accuracy and provide diagnostic and prognostic evaluation of patients with Graves' disease. The present review focuses on the development of TRAbs bioassays, and particularly on the role that Leonard D. Kohn had in this. Indeed, 30 years ago, the Kohn group developed a bioassay based on the use of FRTL-5 cells that was characterized by high reproducibility, feasibility, and diagnostic accuracy. Using this FRTL-5 bioassay, Kohn and his colleagues were the first to develop monoclonal antibodies (moAbs) against the TSHR. Furthermore, they demonstrated the multifaceted functional nature of TRAbs in patients with Graves' disease, with the identification of stimulating and blocking TRAbs, and even antibodies that activated pathways other than cAMP. After the cloning of the TSHR, the Kohn laboratory constructed human TSHR-rat luteinizing hormone/chorionic gonadotropin receptor chimeras. This paved the way to a new bioassay based on the use of non-thyroid cells transfected with the Mc4 chimera. The new Mc4 bioassay is characterized by high diagnostic and prognostic accuracy, greater than for other assays. The availability of a commercial kit based on the Mc4 chimera is spreading the use of this assay worldwide, indicating its benefits for these patients with Graves' disease. This review also describes the main contributions made by other researchers in TSHR molecular biology and TRAbs assay, especially with the development of highly potent moAbs. A comparison of the diagnostic accuracies of the main TRAbs assays, as both immunoassays and bioassays, is also provided.

  17. Distinct roles of synaptic and extrasynaptic GABAA receptors in striatal inhibition dynamics

    Directory of Open Access Journals (Sweden)

    Ruixi eLuo

    2013-11-01

    Full Text Available Striatonigral and striatopallidal projecting medium spiny neurons (MSNs express dopamine D1 (D1+ and D2 receptors (D2+, respectively. Both classes receive extensive GABAergic input via expression of synaptic, perisynaptic and extrasynaptic GABAA receptors. The activation patterns of different presynaptic GABAergic neurons produce transient and sustained GABAA receptor-mediated conductance that fulfill distinct physiological roles. We performed single and dual whole cell recordings from striatal neurons in mice expressing fluorescent proteins in interneurons and MSNs. We report specific inhibitory dynamics produced by distinct activation patterns of presynaptic GABAergic neurons as source of synaptic, perisynaptic and extrasynaptic inhibition. Synaptic GABAA receptors in MSNs contain the α2, γ2 and a β subunit. In addition, there is evidence for the developmental increase of the α1 subunit that contributes to faster inhibitory postsynaptic current (IPSC. Tonic GABAergic currents in MSNs from adult mice are carried by extrasynaptic receptors containing the α4 and δ subunit, while in younger mice this current is mediated by receptors that contain the α5 subunit. Both forms of tonic currents are differentially expressed in D1+ and D2+ MSNs. This study extends these findings by relating presynaptic activation with pharmacological analysis of inhibitory conductance in mice where the β3 subunit is conditionally removed in fluorescently labeled D2+ MSNs and in mice with global deletion of the δ subunit. Our results show that responses to low doses of gaboxadol (2μM, a GABAA receptor agonist with preference to δ subunit, are abolished in the δ but not the β3 subunit knock out mice. This suggests that the β3 subunit is not a component of the adult extrasynaptic receptor pool, in contrast to what has been shown for tonic current in young mice. Deletion of the β3 subunit from D2+ MSNs however, removed slow spontaneous IPSCs, implicating its

  18. The role of GluN2A and GluN2B NMDA receptor subunits in AgRP and POMC neurons on body weight and glucose homeostasis.

    Science.gov (United States)

    Üner, Aykut; Gonçalves, Gabriel H M; Li, Wenjing; Porceban, Matheus; Caron, Nicole; Schönke, Milena; Delpire, Eric; Sakimura, Kenji; Bjørbæk, Christian

    2015-10-01

    Hypothalamic agouti-related peptide (AgRP) and pro-opiomelanocortin (POMC) expressing neurons play critical roles in control of energy balance. Glutamatergic input via n-methyl-d-aspartate receptors (NMDARs) is pivotal for regulation of neuronal activity and is required in AgRP neurons for normal body weight homeostasis. NMDARs typically consist of the obligatory GluN1 subunit and different GluN2 subunits, the latter exerting crucial differential effects on channel activity and neuronal function. Currently, the role of specific GluN2 subunits in AgRP and POMC neurons on whole body energy and glucose balance is unknown. We used the cre-lox system to genetically delete GluN2A or GluN2B only from AgRP or POMC neurons in mice. Mice were then subjected to metabolic analyses and assessment of AgRP and POMC neuronal function through morphological studies. We show that loss of GluN2B from AgRP neurons reduces body weight, fat mass, and food intake, whereas GluN2B in POMC neurons is not required for normal energy balance control. GluN2A subunits in either AgRP or POMC neurons are not required for regulation of body weight. Deletion of GluN2B reduces the number of AgRP neurons and decreases their dendritic length. In addition, loss of GluN2B in AgRP neurons of the morbidly obese and severely diabetic leptin-deficient Lep (ob/ob) mice does not affect body weight and food intake but, remarkably, leads to full correction of hyperglycemia. Lep (ob/ob) mice lacking GluN2B in AgRP neurons are also more sensitive to leptin's anti-obesity actions. GluN2B-containing NMDA receptors in AgRP neurons play a critical role in central control of body weight homeostasis and blood glucose balance via mechanisms that likely involve regulation of AgRP neuronal survival and structure, and modulation of hypothalamic leptin action.

  19. Multiple Taf subunits of TFIID interact with Ino2 activation domains and contribute to expression of genes required for yeast phospholipid biosynthesis.

    Science.gov (United States)

    Hintze, Stefan; Engelhardt, Maike; van Diepen, Laura; Witt, Eric; Schüller, Hans-Joachim

    2017-12-01

    Expression of phospholipid biosynthetic genes in yeast requires activator protein Ino2 which can bind to the UAS element inositol/choline-responsive element (ICRE) and trigger activation of target genes, using two separate transcriptional activation domains, TAD1 and TAD2. However, it is still unknown which cofactors mediate activation by TADs of Ino2. Here, we show that multiple subunits of basal transcription factor TFIID (TBP-associated factors Taf1, Taf4, Taf6, Taf10 and Taf12) are able to interact in vitro with activation domains of Ino2. Interaction was no longer observed with activation-defective variants of TAD1. We were able to identify two nonoverlapping regions in the N-terminus of Taf1 (aa 1-100 and aa 182-250) each of which could interact with TAD1 of Ino2 as well as with TAD4 of activator Adr1. Specific missense mutations within Taf1 domain aa 182-250 affecting basic and hydrophobic residues prevented interaction with wild-type TAD1 and caused reduced expression of INO1. Using chromatin immunoprecipitation we demonstrated Ino2-dependent recruitment of Taf1 and Taf6 to ICRE-containing promoters INO1 and CHO2. Transcriptional derepression of INO1 was no longer possible with temperature-sensitive taf1 and taf6 mutants cultivated under nonpermissive conditions. This result supports the hypothesis of Taf-dependent expression of structural genes activated by Ino2. © 2017 John Wiley & Sons Ltd.

  20. Ketamine-induced inhibition of glycogen synthase kinase-3 contributes to the augmentation of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor signaling.

    Science.gov (United States)

    Beurel, Eléonore; Grieco, Steven F; Amadei, Celeste; Downey, Kimberlee; Jope, Richard S

    2016-09-01

    Sub-anesthetic doses of ketamine have been found to provide rapid antidepressant actions, indicating that the cellular signaling systems targeted by ketamine are potential sites for therapeutic intervention. Ketamine acts as an antagonist of N-methyl-D-aspartate (NMDA) receptors, and animal studies indicate that subsequent augmentation of signaling by α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors is critical for the antidepressant outcome. In this study, we tested if the inhibitory effect of ketamine on glycogen synthase kinase-3 (GSK3) affected hippocampal cell-surface AMPA receptors using immunoblotting of membrane and synaptosomal extracts from wild-type and GSK3 knockin mice. Treatment with an antidepressant dose of ketamine increased the hippocampal membrane level of the AMPA glutamate receptor (GluA)1 subunit, but did not alter the localization of GluA2, GluA3, or GluA4. This effect of ketamine was abrogated in GSK3 knockin mice expressing mutant GSK3 that cannot be inhibited by ketamine, demonstrating that ketamine-induced inhibition of GSK3 is necessary for up-regulation of cell surface AMPA GluA1 subunits. AMPA receptor trafficking is regulated by post-synaptic density-95 (PSD-95), a substrate for GSK3. Ketamine treatment decreased the hippocampal membrane level of phosphorylated PSD-95 on Thr-19, the target of GSK3 that promotes AMPA receptor internalization. These results demonstrate that ketamine-induced inhibition of GSK3 causes reduced phosphorylation of PSD-95, diminishing the internalization of AMPA GluA1 subunits to allow for augmented signaling through AMPA receptors following ketamine treatment. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  1. Cloning of the gene encoding the δ subunit of the human T-cell receptor reveals its physical organization within the α-subunit locus and its involvement in chromosome translocations in T-cell malignancy

    International Nuclear Information System (INIS)

    Isobe, M.; Russo, G.; Haluska, F.G.; Croce, C.M.

    1988-01-01

    By taking advantage of chromosomal walking techniques, the authors have obtained clones that encompass the T-cell receptor (TCR) δ-chain gene. They analyzed clones spanning the entire J α region extending 115 kilobases 5' of the TCR α-chain constant region and have shown that the TCR δ-chain gene is located over 80 kilobases 5' of C α . TCR δ-chain gene is rearranged in the γ/δ-expressing T-cell line Peer and is deleted in α/β-expressing T-cell lines. Sequence analysis of portions of this genomic region demonstrates its identity with previously described cDNA clones corresponding to the C δ and J δ segments. Furthermore, they have analyzed a t(8;14)-(q24;q11) chromosome translocation from a T-cell leukemia and have shown that the J δ segment is rearranged in cells deriving from this tumor and probably directly involved in the translocation. Thus, the newly clones TCR δ chain is implicated in the genesis of chromosome translocations in T-cell malignancies carrying cytogenetic abnormalities of band 14q11

  2. Both point mutations and low expression levels of the nicotinic acetylcholine receptor β1 subunit are associated with imidacloprid resistance in an Aphis gossypii (Glover) population from a Bt cotton field in China.

    Science.gov (United States)

    Chen, Xuewei; Li, Fen; Chen, Anqi; Ma, Kangsheng; Liang, Pingzhuo; Liu, Ying; Song, Dunlun; Gao, Xiwu

    2017-09-01

    Aphis gossypii Glover is a destructive pest of numerous crops throughout the world. Although the expansion of Bt cotton cultivation has helped to control some insect pests, the damage from cotton aphids has not been mitigated. The evolution of aphid resistance to imidacloprid has made its chemical control more difficult since its introduction in 1991. Field populations of A. gossypii that were collected from different transgenic (Bt) cotton planting areas of China in 2014 developed different levels of resistance to imidacloprid. The IMI_R strain has developed high resistance to imidacloprid with the resistance ratio >1200-fold. Compared with the susceptible IMI_S strain, the IMI_R strain also developed a high level cross resistance to sulfoxaflor and acetamiprid. The limited synergism with either PBO or DEF suggests that resistance may be due to the site mutation of molecular target rather than to enhanced detoxification. Three target-site mutations within the nicotinic acetylcholine receptor (nAChR) β1 subunit were detected in the IMI_R strain. The R81T mutation has been reported to be responsible for imidacloprid resistance in A. gossypii and M. persicae. Both V62I and K264E were first detected in A. gossypii. These point mutations are also present in field populations, suggesting that they play a role in the resistance to imidacloprid. Furthermore, the expression level of transcripts encoding β1 subunit was decreased significantly in the IMI_R strain compared with the IMI_S strain, suggesting that both point mutations and the down-regulation of nAChR β1 subunit expression may be involved in the resistance mechanism for imidacloprid in A. gossypii. These results should be useful for the management of imidacloprid-resistant cotton aphids in Bt cotton fields in China. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Motor Skills Training Enhances α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Receptor Subunit mRNA Expression in the Ipsilateral Sensorimotor Cortex and Striatum of Rats Following Intracerebral Hemorrhage.

    Science.gov (United States)

    Tamakoshi, Keigo; Ishida, Kazuto; Kawanaka, Kentaro; Takamatsu, Yasuyuki; Tamaki, Hiroyuki

    2017-10-01

    We investigated the effects of acrobatic training (AT) on expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunits in the sensorimotor cortex and striatum after intracerebral hemorrhage (ICH). Male Wistar rats were divided into 4 groups: ICH without AT (ICH), ICH with AT (ICH + AT), sham operation without AT (SHAM), and sham operation with AT (SHAM + AT). ICH was induced by collagenase injection into the left striatum. The ICH + AT group performed 5 acrobatic tasks daily on days 4-28 post ICH. Forelimb sensorimotor function was evaluated using the forelimb placing test. On days 14 and 29, mRNA expression levels of AMPAR subunits GluR1-4 were measured by real-time reverse transcription-polymerase chain reaction. Forelimb placing test scores were significantly higher in the ICH + AT group than in the ICH group. Expression levels of all AMPAR subunit mRNAs were significantly higher in the ipsilateral sensorimotor cortex of rats in the ICH + AT group than in that of rats in the ICH group on day 29. GluR3 and GluR4 expression levels were reduced in the ipsilateral striatum of rats in the ICH group compared with that of rats in the SHAM group on day 14. These changes may play a critical role in motor skills training-induced recovery after ICH. Copyright © 2017 National Stroke Association. Published by Elsevier Inc. All rights reserved.

  4. Rat intra-hippocampal NMDA infusion induces cell-specific damage and changes in expression of NMDA and GABA(A) receptor subunits

    Czech Academy of Sciences Publication Activity Database

    Rambousek, Lukáš; Kletečková, Lenka; Kubesová, A.; Jirák, D.; Valeš, Karel; Fritschy, J.M.

    2016-01-01

    Roč. 105, Jun (2016), s. 594-606 ISSN 0028-3908 R&D Projects: GA ČR(CZ) GBP304/12/G069; GA ČR(CZ) GA14-20613S; GA ČR(CZ) GAP303/12/1464 Institutional support: RVO:67985823 Keywords : excitotoxicity * NMDA receptor * GABA A receptor * hippocampus * neuroinflammation * neurodegeneration * interneurons * spatial learning * carousel maze Subject RIV: FH - Neurology Impact factor: 5.012, year: 2016

  5. Contribution of N-methyl-D-aspartate receptors to attention and episodic spatial memory during senescence.

    Science.gov (United States)

    Guidi, Michael; Rani, Asha; Karic, Semir; Severance, Barrett; Kumar, Ashok; Foster, Thomas C

    2015-11-01

    A decrease in N-methyl-D-aspartate receptor (NMDAR) function is associated with age-related cognitive impairments. However, NMDAR antagonists are prescribed for cognitive decline associated with age-related neurodegenerative disease, raising questions as to the role of NMDAR activity in cognitive function during aging. The current studies examined effects of NMDAR blockade on cognitive task that are sensitive to aging. Young and middle-age rats were trained on the five-choice serial reaction time task (5-CSRTT) and challenged with MK-801 (0.025, 0.05, and 0.1mg/kg or vehicle). Attention deficits were apparent in middle-age and performance of young and middle-age rats was enhanced for low doses of MK-801 (0.025 and 0.05). The beneficial effects on attention were reversed by the highest dose of MK-801. Older animals exhibited a delay-dependent impairment of episodic spatial memory examined on a delayed-matching to place water maze task. Similarly, a low dose of MK-801 (0.05mg/kg) impaired performance with increasing delay and aged animals were more susceptible to disruption by NMDAR blockade. Despite MK-801 impairment of episodic spatial memory, MK-801 had minimal effects on spatial reference memory. Our results confirm that NMDARs contribute to rapidly acquired and flexible spatial memory and support the idea that a decline in NMDAR function contributes to the age-related impairments in cognition. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. The complement anaphylatoxin C5a receptor contributes to obese adipose tissue inflammation and insulin resistance.

    Science.gov (United States)

    Phieler, Julia; Chung, Kyoung-Jin; Chatzigeorgiou, Antonios; Klotzsche-von Ameln, Anne; Garcia-Martin, Ruben; Sprott, David; Moisidou, Maria; Tzanavari, Theodora; Ludwig, Barbara; Baraban, Elena; Ehrhart-Bornstein, Monika; Bornstein, Stefan R; Mziaut, Hassan; Solimena, Michele; Karalis, Katia P; Economopoulou, Matina; Lambris, John D; Chavakis, Triantafyllos

    2013-10-15

    Obese adipose tissue (AT) inflammation contributes critically to development of insulin resistance. The complement anaphylatoxin C5a receptor (C5aR) has been implicated in inflammatory processes and as regulator of macrophage activation and polarization. However, the role of C5aR in obesity and AT inflammation has not been addressed. We engaged the model of diet-induced obesity and found that expression of C5aR was significantly upregulated in the obese AT, compared with lean AT. In addition, C5a was present in obese AT in the proximity of macrophage-rich crownlike structures. C5aR-sufficient and -deficient mice were fed a high-fat diet (HFD) or a normal diet (ND). C5aR deficiency was associated with increased AT weight upon ND feeding in males, but not in females, and with increased adipocyte size upon ND and HFD conditions in males. However, obese C5aR(-/-) mice displayed improved systemic and AT insulin sensitivity. Improved AT insulin sensitivity in C5aR(-/-) mice was associated with reduced accumulation of total and proinflammatory M1 macrophages in the obese AT, increased expression of IL-10, and decreased AT fibrosis. In contrast, no difference in β cell mass was observed owing to C5aR deficiency under an HFD. These results suggest that C5aR contributes to macrophage accumulation and M1 polarization in the obese AT and thereby to AT dysfunction and development of AT insulin resistance.

  7. [Contributions of neuropsychology to anti-NMDA receptor antibody encephalitis: a literature review].

    Science.gov (United States)

    Luna-Lario, P; Hernaez-Goni, P; Tirapu-Ustarroz, J

    2016-05-01

    Limbic encephalitis generated by anti-N-methyl-D-aspartate (NMDA) receptor antibodies is an acute and severe neurological entity, which is more prevalent in young females and is associated to an underlying tumour. Since it leads to severe cognitive impairment, thought needs to be given to the contributions of neuropsychology to the diagnosis, development and treatment of the disease, which have received little attention from researchers to date. A review is conducted of the prior literature, evaluating the measurement of the cognitive symptoms (predominantly mnemonic and executive) associated to this disease. Valid, reliable neuropsychological instruments are proposed, and it is suggested that neuropsychological measures may be used as parameters to follow up these patients which help monitor their functionality in daily living once they have recovered from the acute phase. Similarly they can become a basis on which to assemble rehabilitation programmes that favour the accomplishment of personal autonomy and the patients' reintegration in the community. Nevertheless, we stress the need to include neuropsychologists and neuropsychiatrists in not only the detection but also the treatment of these patients so as to enable them to recover their personal independence and re-adapt to their natural settings.

  8. Source contribution analysis of surface particulate polycyclic aromatic hydrocarbon concentrations in northeastern Asia by source–receptor relationships

    International Nuclear Information System (INIS)

    Inomata, Yayoi; Kajino, Mizuo; Sato, Keiichi; Ohara, Toshimasa; Kurokawa, Jun-ichi; Ueda, Hiromasa; Tang, Ning; Hayakawa, Kazuichi; Ohizumi, Tsuyoshi; Akimoto, Hajime

    2013-01-01

    We analyzed the source–receptor relationships for particulate polycyclic aromatic hydrocarbon (PAH) concentrations in northeastern Asia using an aerosol chemical transport model. The model successfully simulated the observed concentrations. In Beijing (China) benzo[a]pyren (BaP) concentrations are due to emissions from its own domain. In Noto, Oki and Tsushima (Japan), transboundary transport from northern China (>40°N, 40–60%) and central China (30–40°N, 10–40%) largely influences BaP concentrations from winter to spring, whereas the relative contribution from central China is dominant (90%) in Hedo. In the summer, the contribution from Japanese domestic sources increases (40–80%) at the 4 sites. Contributions from Japan and Russia are additional source of BaP over the northwestern Pacific Ocean in summer. The contribution rates for the concentrations from each domain are different among PAH species depending on their particulate phase oxidation rates. Reaction with O 3 on particulate surfaces may be an important component of the PAH oxidation processes. -- Highlights: •Source–receptor analysis was conducted for investigating PAHs in northeast Asia. •In winter, transboundary transport from China is large contribution in leeward. •Relative contribution from Korea, Japan, and eastern Russia is increased in summer. •This seasonal variation is strongly controlled by the meteorological conditions. •The transport distance is different among PAH species. -- Transboundary transport of PAHs in northeast Asia was investigated by source–receptor analysis

  9. Targeted deletion of the GABRA2 gene encoding alpha2-subunits of GABA(A) receptors facilitates performance of a conditioned emotional response, and abolishes anxiolytic effects of benzodiazepines and barbiturates.

    Science.gov (United States)

    Dixon, C I; Rosahl, T W; Stephens, D N

    2008-07-01

    Mice with point-mutated alpha2 GABA(A) receptor subunits (rendering them diazepam insensitive) are resistant to the anxiolytic-like effects of benzodiazepines (BZs) in the conditioned emotional response (CER) test, but show normal anxiolytic effects of a barbiturate. We investigated the consequence of deleting the alpha2-subunit on acquisition of the CER with increasing intensity of footshock, and on the anxiolytic efficacy of a benzodiazepine, diazepam, and a barbiturate, pentobarbital. alpha2 knockout (KO) and wildtype (WT) mice were trained in a conditioned emotional response (CER) task, in which lever pressing for food on a variable interval (VI) schedule was suppressed during the presentation of a compound light/tone conditioned stimulus (CS+) that predicted footshock. The ability of diazepam and of pentobarbital to reduce suppression during the CS+ was interpreted as an anxiolytic response. There were no differences between the genotypes in shock sensitivity, as assessed by their flinch responses to increasing levels of shock. However, alpha2 KO mice showed a greater suppression of lever pressing than WT littermates in the presence of a compound cue signalling footshock. Diazepam (0, 0.5, 1 and 2 mg/kg) induced a dose-dependent anxiolytic-like effect in WT mice but no such effect was seen in KO mice. Similarly, although pentobarbital (20 mg/kg) reduced the ability of the CS+ to reduce lever pressing rates in WT mice, this effect was not seen in the KO. These findings suggest that alpha2-containing GABA(A) receptors mediate the anxiolytic effects of barbiturates, as well as benzodiazepines, and that they may be involved in neuronal circuits underlying conditioned anxiety.

  10. Minute Impurities Contribute Significantly to Olfactory Receptor Ligand Studies: Tales from Testing the Vibration Theory

    OpenAIRE

    Paoli, M.; M?nch, D.; Haase, A.; Skoulakis, E.; Turin, L.; Galizia, C. G.

    2017-01-01

    Several studies have attempted to test the vibrational hypothesis of odorant receptor activation in behavioral and physiological studies using deuterated compounds as odorants. The results have been mixed. Here, we attempted to test how deuterated compounds activate odorant receptors using calcium imaging of the fruit fly antennal lobe. We found specific activation of one area of the antennal lobe corresponding to inputs from a specific receptor. However, upon more detailed analysis, we disco...

  11. The Fas/Fas ligand death receptor pathway contributes to phenylalanine-induced apoptosis in cortical neurons.

    Directory of Open Access Journals (Sweden)

    Xiaodong Huang

    Full Text Available Phenylketonuria (PKU, an autosomal recessive disorder of amino acid metabolism caused by mutations in the phenylalanine hydroxylase (PAH gene, leads to childhood mental retardation by exposing neurons to cytotoxic levels of phenylalanine (Phe. A recent study showed that the mitochondria-mediated (intrinsic apoptotic pathway is involved in Phe-induced apoptosis in cultured cortical neurons, but it is not known if the death receptor (extrinsic apoptotic pathway and endoplasmic reticulum (ER stress-associated apoptosis also contribute to neurodegeneration in PKU. To answer this question, we used specific inhibitors to block each apoptotic pathway in cortical neurons under neurotoxic levels of Phe. The caspase-8 inhibitor Z-IETD-FMK strongly attenuated apoptosis in Phe-treated neurons (0.9 mM, 18 h, suggesting involvement of the Fas receptor (FasR-mediated cell death receptor pathway in Phe toxicity. In addition, Phe significantly increased cell surface Fas expression and formation of the Fas/FasL complex. Blocking Fas/FasL signaling using an anti-Fas antibody markedly inhibited apoptosis caused by Phe. In contrast, blocking the ER stress-induced cell death pathway with salubrinal had no effect on apoptosis in Phe-treated cortical neurons. These experiments demonstrate that the Fas death receptor pathway contributes to Phe-induced apoptosis and suggest that inhibition of the death receptor pathway may be a novel target for neuroprotection in PKU patients.

  12. Upregulation of calcium channel alpha-2-delta-1 subunit in dorsal horn contributes to spinal cord injury-induced tactile allodynia.

    Science.gov (United States)

    Kusuyama, Kazuki; Tachibana, Toshiya; Yamanaka, Hiroki; Okubo, Masamichi; Yoshiya, Shinichi; Noguchi, Koichi

    2018-01-31

    Spinal cord injury (SCI) commonly results not only in motor paralysis but also in the emergence of neuropathic pain (NeuP), both of which can impair the quality of life for patients with SCI. In the clinical field, it is well known that pregabalin, which binds to the voltage-gated calcium channel alpha-2-delta-1 (α 2 δ-1) subunit has therapeutic effects on NeuP after SCI. A previous study has demonstrated that SCI increased α 2 δ-1 in the L4-L6 dorsal spinal cord of SCI rats by Western blot analysis and that the increase of α 2 δ-1 was correlated with tactile allodynia of the hind paw. However, the detailed feature of an increase in α 2 δ-1 protein in the spinal dorsal horn and the mechanism of pregabalin effect on SCI-induced NeuP have not been fully examined. This study aimed to examine the detailed distribution of α 2 δ-1 expression in the lumbar spinal cord after thoracic SCI in rats and the correlation of the therapeutic effect of pregabalin in SCI rats. Male Sprague-Dawley rats underwent thoracic (T10) spinal cord contusion injury using the IH impactor device. Spinal cord injury rats received pregabalin (30 mg/kg) once a day for 2 weeks over a 4-week period after SCI. The mechanical threshold in the rat hind paw was measured over 4 weeks. Alpha-2-delta-1 expression in the lumbar spinal cord and in the dorsal root ganglion (DRG) was analyzed using immunohistochemistry and in situ hybridization histochemistry. A significant reduction of the withdrawal threshold of mechanical stimuli to the hind paw was observed for 2 weeks and continued at least 4 weeks after SCI. In the control rats, expression of α 2 δ-1 immunoreactivity was detected mainly in laminae I and II in the lumbar dorsal horn. Thoracic SCI significantly increased α 2 δ-1 immunoreactivity in laminae I and II in the lumbar dorsal horn 4 weeks after SCI; however, thoracic SCI did not affect the expression of α 2 δ-1 mRNA in the L4 and L5 DRGs. Meanwhile, the signal intensity of α 2

  13. Identification of transmembrane domain 6 & 7 residues that contribute to the binding pocket of the urotensin II receptor.

    Science.gov (United States)

    Holleran, Brian J; Domazet, Ivana; Beaulieu, Marie-Eve; Yan, Li Ping; Guillemette, Gaétan; Lavigne, Pierre; Escher, Emanuel; Leduc, Richard

    2009-04-15

    Urotensin II (U-II), a cyclic undecapeptide, is the natural ligand of the urotensin II (UT) receptor, a G protein-coupled receptor. In the present study, we used the substituted-cysteine accessibility method to identify specific residues in transmembrane domains (TMDs) six and seven of the rat urotensin II receptor (rUT) that contribute to the formation of the binding pocket of the receptor. Each residue in the R256(6.32)-Q283(6.59) fragment of TMD6 and the A295(7.31)-T321(7.57) fragment of TMD7 was mutated, individually, to a cysteine. The resulting mutants were expressed in COS-7 cells, which were subsequently treated with the positively charged methanethiosulfonate-ethylammonium (MTSEA) or the negatively charged methanethiosulfonate-ethylsulfonate (MTSES) sulfhydryl-specific alkylating agents. MTSEA treatment resulted in a significant reduction in the binding of TMD6 mutants F268C(6.44) and W278C(6.54) and TMD7 mutants L298C(7.34), T302C(7.38), and T303C(7.39) to (125)I-U-II. MTSES treatment resulted in a significant reduction in the binding of two additional mutants, namely L282C(6.58) in TMD6 and Y300C(7.36) in TMD7. These results suggest that specific residues orient themselves within the water-accessible binding pocket of the rUT receptor. This approach, which allowed us to identify key determinants in TMD6 and TMD7 that contribute to the UT receptor binding pocket, enabled us to further refine our homology-based model of how U-II interacts with its cognate receptor.

  14. Dopamine D(3) receptors contribute to methamphetamine-induced alterations in dopaminergic neuronal function: role of hyperthermia.

    Science.gov (United States)

    Baladi, Michelle G; Newman, Amy H; Nielsen, Shannon M; Hanson, Glen R; Fleckenstein, Annette E

    2014-06-05

    Methamphetamine administration causes long-term deficits to dopaminergic systems that, in humans, are thought to be associated with motor slowing and memory impairment. Methamphetamine interacts with the dopamine transporter (DAT) and increases extracellular concentrations of dopamine that, in turn, binds to a number of dopamine receptor subtypes. Although the relative contribution of each receptor subtype to the effects of methamphetamine is not fully known, non-selective dopamine D2/D3 receptor antagonists can attenuate methamphetamine-induced changes to dopamine systems. The present study extended these findings by testing the role of the dopamine D3 receptor subtype in mediating the long-term dopaminergic, and for comparison serotonergic, deficits caused by methamphetamine. Results indicate that the dopamine D3 receptor selective antagonist, PG01037, attenuated methamphetamine-induced decreases in striatal DAT, but not hippocampal serotonin (5HT) transporter (SERT), function, as assessed 7 days after treatment. However, PG01037 also attenuated methamphetamine-induced hyperthermia. When methamphetamine-induced hyperthermia was maintained by treating rats in a warm ambient environment, PG01037 failed to attenuate the effects of methamphetamine on DAT uptake. Furthermore, PG01037 did not attenuate methamphetamine-induced decreases in dopamine and 5HT content. Taken together, the present study demonstrates that dopamine D3 receptors mediate, in part, the long-term deficits in DAT function caused by methamphetamine, and that this effect likely involves an attenuation of methamphetamine-induced hyperthermia. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Dopamine D3 receptors contribute to methamphetamine-induced alterations in dopaminergic neuronal function: Role of hyperthermia

    Science.gov (United States)

    Baladi, Michelle G.; Newman, Amy H.; Nielsen, Shannon M.; Hanson, Glen R.; Fleckenstein, Annette E.

    2014-01-01

    Methamphetamine administration causes long-term deficits to dopaminergic systems that, in humans, are thought to be associated with motor slowing and memory impairment. Methamphetamine interacts with the dopamine transporter (DAT) and increases extracellular concentrations of dopamine that, in turn, binds to a number of dopamine receptor subtypes. Although the relative contribution of each receptor subtype to the effects of methamphetamine is not fully known, non-selective dopamine D2/D3 receptor antagonists can attenuate methamphetamine-induced changes to dopamine systems. The present study extended these findings by testing the role of the dopamine D3 receptor subtype in mediating the long-term dopaminergic, and for comparison serotonergic, deficits caused by methamphetamine. Results indicate that the dopamine D3 receptor selective antagonist, PG01037, attenuated methamphetamine-induced decreases in striatal DAT, but not hippocampal serotonin (5HT) transporter (SERT), function, as assessed 7 days after treatment. However, PG01037 also attenuated methamphetamine-induced hyperthermia. When methamphetamine-induced hyperthermia was maintained by treating rats in a warm ambient environment, PG01037 failed to attenuate the effects of methamphetamine on DAT uptake. Furthermore, PG01037 did not attenuate methamphetamine-induced decreases in dopamine and 5HT content. Taken together, the present study demonstrates that dopamine D3 receptors mediate, in part, the long-term deficits in DAT function caused by methamphetamine, and that this effect likely involves an attenuation of methamphetamine-induced hyperthermia. PMID:24685638

  16. Nerve terminal contributes to acetylcholine receptor organization at the dystrophic neuromuscular junction of mdx mice.

    Science.gov (United States)

    Marques, Maria Julia; Taniguti, Ana Paula Tiemi; Minatel, Elaine; Neto, Humberto Santo

    2007-02-01

    Changes in the distribution of acetylcholine receptors have been reported to occur at the neuromuscular junction of mdx mice and may be a consequence of muscle fiber regeneration rather than the absence of dystrophin. In the present study, we examined whether the nerve terminal determines the fate of acetylcholine receptor distribution in the dystrophic muscle fibers of mdx mice. The left sternomastoid muscle of young (1-month-old) and adult (6-month-old) mdx mice was injected with 60 microl lidocaine hydrochloride to induce muscle degeneration-regeneration. Some mice had their sternomastoid muscle denervated at the time of lidocaine injection. After 10 days of muscle denervation, nerve terminals and acetylcholine receptors were labeled with 4-Di-2-ASP and rhodamine-alpha-bungarotoxin, respectively, for confocal microscopy. In young mdx mice, 75% (n = 137 endplates) of the receptors were distributed in islands. The same was observed in 100% (n = 114 endplates) of the adult junctions. In denervated-regenerated fibers of young mice, the receptors were distributed as branches in 89% of the endplates (n = 90). In denervated-regenerated fibers of adult mice, the receptors were distributed in islands in 100% of the endplates (n = 100). These findings show that nerve-dependent mechanisms are also involved in the changes in receptor distribution in young dystrophic muscles. In older dystrophic muscles, other factors may play a role in receptor distribution.

  17. Structure-Function Evaluation of Imidazopyridine Derivatives Selective for δ-Subunit-Containing γ-Aminobutyric Acid Type A (GABAA) Receptors.

    Science.gov (United States)

    Yakoub, Kirsten; Jung, Sascha; Sattler, Christian; Damerow, Helen; Weber, Judith; Kretzschmann, Annika; Cankaya, Aylin S; Piel, Markus; Rösch, Frank; Haugaard, Anne S; Frølund, Bente; Schirmeister, Tanja; Lüddens, Hartmut

    2018-03-08

    δ-Selective compounds 1 and 2 (DS1, compound 22; DS2, compound 16) were introduced as functionally selective modulators of δ-containing GABA type A receptors (GABA A R). In our hands, [ 3 H]EBOB-binding experiments with recombinant GABA A R and compound 22 showed no proof of δ-selectivity, although there was a minimally higher preference for the α4β3δ and α6β2/3δ receptors with respect to potency. In order to delineate the structural determinants of δ preferences, we synthesized 25 derivatives of DS1 and DS2, and investigated their structure-activity relationships (SAR). Four of our derivatives showed selectivity for α6β3δ receptors (29, 38, 39, and 41). For all of them, the major factors that distinguished them from compound 22 were variations at the para-positions of their benzamide groups. However, two compounds (29 and 39), when tested in the presence of GABA, revealed effects at several additional GABA A R. The newly synthesized compounds will still serve as useful tools to investigate α6β3δ receptors.

  18. Double Dissociation of Spike Timing-Dependent Potentiation and Depression by Subunit-Preferring NMDA Receptor Antagonists in Mouse Barrel Cortex

    NARCIS (Netherlands)

    Banerjee, A.; Meredith, R.M.; Rodriguez-Moreno, A.; Mierau, S.B.; Auberson, Y.P.; Paulsen, O.

    2009-01-01

    Spike timing-dependent plasticity (STDP) is a strong candidate for an N-methyl-D-aspartate (NMDA) receptor-dependent form of synaptic plasticity that could underlie the development of receptive field properties in sensory neocortices. Whilst induction of timing-dependent long-term potentiation

  19. Transcriptional regulators of Na, K-ATPase subunits

    OpenAIRE

    Zhiqin eLi; Sigrid A Langhans

    2015-01-01

    The Na,K-ATPase classically serves as an ion pump creating an electrochemical gradient across the plasma membrane that is essential for transepithelial transport, nutrient uptake and membrane potential. In addition, Na,K-ATPase also functions as a receptor, a signal transducer and a cell adhesion molecule. With such diverse roles, it is understandable that the Na,K-ATPase subunits, the catalytic alpha-subunit, the beta-subunit and the FXYD proteins, are controlled extensively during developme...

  20. Potential contribution of progesterone receptors to the development of sexual behavior in male and female mice.

    Science.gov (United States)

    Desroziers, Elodie; Brock, Olivier; Bakker, Julie

    2017-04-01

    We previously showed that estradiol can have both defeminizing and feminizing effects on the developing mouse brain. Pre- and early postnatal estradiol defeminized the ability to show lordosis in adulthood, whereas prepubertal estradiol feminized this ability. Furthermore, we found that estradiol upregulates progesterone receptors (PR) during development, inducing both a male-and female-typical pattern of PR expression in the mouse hypothalamus. In the present study, we took advantage of a newly developed PR antagonist (ZK 137316) to determine whether PR contributes to either male- or female-typical sexual differentiation. Thus groups of male and female C57Bl/6j mice were treated with ZK 137316 or OIL as control: males were treated neonatally (P0-P10), during the critical period for male sexual differentiation, and females were treated prepubertally (P15-P25), during the critical period for female sexual differentiation. In adulthood, mice were tested for sexual behavior. In males, some minor effects of neonatal ZK treatment on sexual behavior were observed: latencies to the first mount, intromission and ejaculation were decreased in neonatally ZK treated males; however, this effect disappeared by the second mating test. By contrast, female mice treated with ZK during the prepubertal period showed significantly less lordosis than OIL-treated females. Mate preferences were not affected in either males or females treated with ZK during development. Taken together, these results suggest a role for PR and thus perhaps progesterone in the development of lordosis behavior in female mice. By contrast, no obvious role for PR can be discerned in the development of male sexual behavior. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Altered learning, memory, and social behavior in type 1 taste receptor subunit 3 knock-out mice are associated with neuronal dysfunction.

    Science.gov (United States)

    Martin, Bronwen; Wang, Rui; Cong, Wei-Na; Daimon, Caitlin M; Wu, Wells W; Ni, Bin; Becker, Kevin G; Lehrmann, Elin; Wood, William H; Zhang, Yongqing; Etienne, Harmonie; van Gastel, Jaana; Azmi, Abdelkrim; Janssens, Jonathan; Maudsley, Stuart

    2017-07-07

    The type 1 taste receptor member 3 (T1R3) is a G protein-coupled receptor involved in sweet-taste perception. Besides the tongue, the T1R3 receptor is highly expressed in brain areas implicated in cognition, including the hippocampus and cortex. As cognitive decline is often preceded by significant metabolic or endocrinological dysfunctions regulated by the sweet-taste perception system, we hypothesized that a disruption of the sweet-taste perception in the brain could have a key role in the development of cognitive dysfunction. To assess the importance of the sweet-taste receptors in the brain, we conducted transcriptomic and proteomic analyses of cortical and hippocampal tissues isolated from T1R3 knock-out (T1R3KO) mice. The effect of an impaired sweet-taste perception system on cognition functions were examined by analyzing synaptic integrity and performing animal behavior on T1R3KO mice. Although T1R3KO mice did not present a metabolically disrupted phenotype, bioinformatic interpretation of the high-dimensionality data indicated a strong neurodegenerative signature associated with significant alterations in pathways involved in neuritogenesis, dendritic growth, and synaptogenesis. Furthermore, a significantly reduced dendritic spine density was observed in T1R3KO mice together with alterations in learning and memory functions as well as sociability deficits. Taken together our data suggest that the sweet-taste receptor system plays an important neurotrophic role in the extralingual central nervous tissue that underpins synaptic function, memory acquisition, and social behavior. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Evaluation of [methyl-3H]L655,708 and [ethyl-3H]RY80 as putative PET ligands for central GABAA receptors containing α5 subunit

    International Nuclear Information System (INIS)

    Opacka-Juffry, J.; Hirani, E.; Dawson, G.R.; Luthra, S.K.; Hume, S.P.

    1999-01-01

    Two selective radioligands of gamma aminobutyric acid (GABA) A receptors containing the α5 subunit, [ 3 H]L655,708 and [ 3 H]RY80, were evaluated in rats as potential in vivo tracers for positron emission tomography (PET). Brain uptake index (BUI), a measure of first pass extraction, was moderate for [ 3 H]L655,708 (BUI of 59%) and good for [ 3 H]RY80 (BUI of 96%). This finding was consistent with their in vitro binding to plasma proteins of ∼76% and 50%, respectively. Following intravenous injection of either radioligand, radioactivity in plasma was measured and uptake characteristics were assessed in brain within a time period relevant to PET scanning (up to 90 min). Discrete brain regions, such as frontal cortex, striatum, hypothalamus, thalamus, hippocampus, colliculi, medulla, and cerebellum, were sampled and the temporal distribution of radioactivity analysed. Despite the reasonable delivery to the brain, neither of the radioligands had sufficient retention in the tissues rich in α5-containing GABA A receptors to achieve a good selective signal. For both radioligands, a maximal tissue:cerebellum ratio of 1.5 was seen in hippocampus at 10 min after injection. Thus, neither of the compounds studied shows potential for further development as an in vivo PET ligand

  3. Role of the Rubisco Small Subunit

    Energy Technology Data Exchange (ETDEWEB)

    Spreitzer, Robert Joseph [Univ. of Nebraska, Lincoln, NE (United States)

    2016-11-05

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of CO2 fixation in photosynthesis. However, it is a slow enzyme, and O2 competes with CO2 at the active site. Oxygenation initiates the photorespiratory pathway, which also results in the loss of CO2. If carboxylation could be increased or oxygenation decreased, an increase in net CO2 fixation would be realized. Because Rubisco provides the primary means by which carbon enters all life on earth, there is much interest in engineering Rubisco to increase the production of food and renewable energy. Rubisco is located in the chloroplasts of plants, and it is comprised of two subunits. Much is known about the chloroplast-gene-encoded large subunit (rbcL gene), which contains the active site, but much less is known about the role of the nuclear-gene-encoded small subunit in Rubisco function (rbcS gene). Both subunits are coded by multiple genes in plants, which makes genetic engineering difficult. In the eukaryotic, green alga Chlamydomonas reinhardtii, it has been possible to eliminate all the Rubisco genes. These Rubisco-less mutants can be maintained by providing acetate as an alternative carbon source. In this project, focus has been placed on determining whether the small subunit might be a better genetic-engineering target for improving Rubisco. Analysis of a variable-loop structure (βA-βB loop) of the small subunit by genetic selection, directed mutagenesis, and construction of chimeras has shown that the small subunit can influence CO2/O2 specificity. X-ray crystal structures of engineered chimeric-loop enzymes have indicated that additional residues and regions of the small subunit may also contribute to Rubisco function. Structural dynamics of the small-subunit carboxyl terminus was also investigated. Alanine-scanning mutagenesis of the most-conserved small-subunit residues has identified a

  4. Two N-glycosylation Sites in the GluN1 Subunit Are Essential for Releasing N-methyl-D-aspartate (NMDA) Receptors from the Endoplasmic Reticulum

    Czech Academy of Sciences Publication Activity Database

    Lichnerová, Katarina; Kaniaková, Martina; Park, S. P.; Skřenková, Kristýna; Wang, Y.- X.; Petralia, R. S.; Suh, Y. H.; Horák, Martin

    2015-01-01

    Roč. 290, č. 30 (2015), s. 18379-18390 ISSN 0021-9258 R&D Projects: GA ČR(CZ) GA14-02219S; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:67985823 Keywords : peptide-N-glycosidase * NMDAR * NMDA receptor * endoplasmic reticulum Subject RIV: FH - Neurology Impact factor: 4.258, year: 2015

  5. STEP activation by Gαq coupled GPCRs opposes Src regulation of NMDA receptors containing the GluN2A subunit

    Science.gov (United States)

    Tian, Meng; Xu, Jian; Lei, Gang; Lombroso, Paul J.; Jackson, Michael F.; MacDonald, John F.

    2016-01-01

    N-methyl-D-aspartate receptors (NMDARs) are necessary for the induction of synaptic plasticity and for the consolidation of learning and memory. NMDAR function is tightly regulated by functionally opposed families of kinases and phosphatases. Herein we show that the striatal-enriched protein tyrosine phosphatase (STEP) is recruited by Gαq-coupled receptors, including the M1 muscarinic acetylcholine receptor (M1R), and opposes the Src tyrosine kinase-mediated increase in the function of NMDARs composed of GluN2A. STEP activation by M1R stimulation requires IP3Rs and can depress NMDA-evoked currents with modest intracellular Ca2+ buffering. Src recruitment by M1R stimulation requires coincident NMDAR activation and can augment NMDA-evoked currents with high intracellular Ca2+ buffering. Our findings suggest that Src and STEP recruitment is contingent on differing intracellular Ca2+ dynamics that dictate whether NMDAR function is augmented or depressed following M1R stimulation. PMID:27857196

  6. Cross talk between AT1 receptors and Toll-like receptor 4 in microglia contributes to angiotensin II-derived ROS production in the hypothalamic paraventricular nucleus.

    Science.gov (United States)

    Biancardi, Vinicia Campana; Stranahan, Alexis M; Krause, Eric G; de Kloet, Annette D; Stern, Javier E

    2016-02-01

    ANG II is thought to increase sympathetic outflow by increasing oxidative stress and promoting local inflammation in the paraventricular nucleus (PVN) of the hypothalamus. However, the relative contributions of inflammation and oxidative stress to sympathetic drive remain poorly understood, and the underlying cellular and molecular targets have yet to be examined. ANG II has been shown to enhance Toll-like receptor (TLR)4-mediated signaling on microglia. Thus, in the present study, we aimed to determine whether ANG II-mediated activation of microglial TLR4 signaling is a key molecular target initiating local oxidative stress in the PVN. We found TLR4 and ANG II type 1 (AT1) receptor mRNA expression in hypothalamic microglia, providing molecular evidence for the potential interaction between these two receptors. In hypothalamic slices, ANG II induced microglial activation within the PVN (∼65% increase, P receptors and TLR4 in mediating ANG II-dependent microglial activation and oxidative stress within the PVN. More broadly, our results support a functional interaction between the central renin-angiotensin system and innate immunity in the regulation of neurohumoral outflows from the PVN. Copyright © 2016 the American Physiological Society.

  7. Adenosine A1 receptors contribute to immune regulation after neonatal hypoxic ischemic brain injury

    OpenAIRE

    Winerdal, Max; Winerdal, Malin E.; Wang, Ying-Qing; Fredholm, Bertil B.; Winqvist, Ola; Ådén, Ulrika

    2015-01-01

    Neonatal brain hypoxic ischemia (HI) often results in long-term motor and cognitive impairments. Post-ischemic inflammation greatly effects outcome and adenosine receptor signaling modulates both HI and immune cell function. Here, we investigated the influence of adenosine A1 receptor deficiency (A1R−/−) on key immune cell populations in a neonatal brain HI model. Ten-day-old mice were subjected to HI. Functional outcome was assessed by open locomotion and beam walking test and infarction siz...

  8. Traxoprodil, a selective antagonist of the NR2B subunit of the NMDA receptor, potentiates the antidepressant-like effects of certain antidepressant drugs in the forced swim test in mice.

    Science.gov (United States)

    Poleszak, Ewa; Stasiuk, Weronika; Szopa, Aleksandra; Wyska, Elżbieta; Serefko, Anna; Oniszczuk, Anna; Wośko, Sylwia; Świąder, Katarzyna; Wlaź, Piotr

    2016-08-01

    One of the newest substances, whose antidepressant activity was shown is traxoprodil, which is a selective antagonist of the NR2B subunit of the NMDA receptor. The main goal of the present study was to evaluate the effect of traxoprodil on animals' behavior using the forced swim test (FST), as well as the effect of traxoprodil (10 mg/kg) on the activity of antidepressants, such as imipramine (15 mg/kg), fluoxetine (5 mg/kg), escitalopram (2 mg/kg) and reboxetine (2.5 mg/kg). Serotonergic lesion and experiment using the selective agonists of serotonin receptors 5-HT1A and 5-HT2 was conducted to evaluate the role of the serotonergic system in the antidepressant action of traxoprodil. Brain concentrations of tested agents were determined using HPLC. The results showed that traxoprodil at a dose of 20 and 40 mg/kg exhibited antidepressant activity in the FST and it was not related to changes in animals' locomotor activity. Co-administration of traxoprodil with imipramine, fluoxetine or escitalopram, each in subtherapeutic doses, significantly affected the animals' behavior in the FST and, what is important, these changes were not due to the severity of locomotor activity. The observed effect of traxoprodil is only partially associated with serotonergic system and is independent of the effect on the 5-HT1A and 5-HT2 serotonin receptors. The results of an attempt to assess the nature of the interaction between traxoprodil and the tested drugs show that in the case of joint administration of traxoprodil and fluoxetine, imipramine or escitalopram, there were interactions in the pharmacokinetic phase.

  9. Contributions of pocket depth and electrostatic interactions to affinity and selectivity of receptors for methylated lysine in water.

    Science.gov (United States)

    Beaver, Joshua E; Peacor, Brendan C; Bain, Julianne V; James, Lindsey I; Waters, Marcey L

    2015-03-21

    Dynamic combinatorial chemistry was used to generate a set of receptors for peptides containing methylated lysine (KMen, n = 0-3) and study the contribution of electrostatic effects and pocket depth to binding affinity and selectivity. We found that changing the location of a carboxylate resulted in an increase in preference for KMe2, presumably based on ability to form a salt bridge with KMe2. The number of charged groups on either the receptor or peptide guest systematically varied the binding affinities to all guests by approximately 1-1.5 kcal mol(-1), with little influence on selectivity. Lastly, formation of a deeper pocket led to both increased affinity and selectivity for KMe3 over the lower methylation states. From these studies, we identified that the tightest binder was a receptor with greater net charge, with a Kd of 0.2 μM, and the receptor with the highest selectivity was the one with the deepest pocket, providing 14-fold selectivity between KMe3 and KMe2 and a Kd for KMe3 of 0.3 μM. This work provides key insights into approaches to improve binding affinity and selectivity in water, while also demonstrating the versatility of dynamic combinatorial chemistry for rapidly exploring the impact of subtle changes in receptor functionality on molecular recognition in water.

  10. Minute Impurities Contribute Significantly to Olfactory Receptor Ligand Studies: Tales from Testing the Vibration Theory.

    Science.gov (United States)

    Paoli, M; Münch, D; Haase, A; Skoulakis, E; Turin, L; Galizia, C G

    2017-01-01

    Several studies have attempted to test the vibrational hypothesis of odorant receptor activation in behavioral and physiological studies using deuterated compounds as odorants. The results have been mixed. Here, we attempted to test how deuterated compounds activate odorant receptors using calcium imaging of the fruit fly antennal lobe. We found specific activation of one area of the antennal lobe corresponding to inputs from a specific receptor. However, upon more detailed analysis, we discovered that an impurity of 0.0006% ethyl acetate in a chemical sample of benzaldehyde-d 5 was entirely responsible for a sizable odorant-evoked response in Drosophila melanogaster olfactory receptor cells expressing dOr42b. Without gas chromatographic purification within the experimental setup, this impurity would have created a difference in the responses of deuterated and nondeuterated benzaldehyde, suggesting that dOr42b be a vibration sensitive receptor, which we show here not to be the case. Our results point to a broad problem in the literature on use of non-GC-pure compounds to test receptor selectivity, and we suggest how the limitations can be overcome in future studies.

  11. Downregulation of DJ-1 Fails to Protect Mitochondrial Complex I Subunit NDUFS3 in the Testes and Contributes to the Asthenozoospermia

    Directory of Open Access Journals (Sweden)

    Yupeng Wang

    2018-01-01

    Full Text Available Asthenozoospermia (AS, an important cause of male infertility, is characterized by reduced sperm motility. Among the aetiologies of AS, inflammation seems to be the main cause. DJ-1, a conserved protein product of the PARK7 gene, is associated with male infertility and plays a role in oxidative stress and inflammation. Although our previous studies showed that a reduction in DJ-1 was accompanied by mitochondrial dysfunction in the sperm of patients with AS, the specific mechanism underlying this association remained unclear. In this study, we found that compared to the patients without AS, the expression of mitochondrial protein nicotinamide adenine dinucleotide dehydrogenase (ubiquinone Fe-S protein 3 (NDUFS3 was also significantly decreased in the sperm of patients with AS. Similarly, decreased expression of DJ-1 and NDUFS3 and reduced mitochondria complex I activity were evident in a rat model of AS. Moreover, we showed that the interaction between DJ-1 and NDUFS3 in rat testes was weakened by ORN treatment. These results suggest that the impaired mitochondrial activity could be due to the broken interaction between DJ-1 and NDUFS3 and that downregulation of DJ-1 in sperm and testes contributes to AS pathogenesis.

  12. Pharmacological characterisation of murine α4β1δ GABAA receptors expressed in Xenopus oocytes

    DEFF Research Database (Denmark)

    Villumsen, Inge S; Wellendorph, Petrine; Smart, Trevor G

    2015-01-01

    BACKGROUND: GABAA receptor subunit composition has a profound effect on the receptor's physiological and pharmacological properties. The receptor β subunit is widely recognised for its importance in receptor assembly, trafficking and post-translational modifications, but its influence on extrasyn......BACKGROUND: GABAA receptor subunit composition has a profound effect on the receptor's physiological and pharmacological properties. The receptor β subunit is widely recognised for its importance in receptor assembly, trafficking and post-translational modifications, but its influence...

  13. Mutation of a zinc-binding residue in the glycine receptor α1 subunit changes ethanol sensitivity in vitro and alcohol consumption in vivo.

    Science.gov (United States)

    McCracken, Lindsay M; Blednov, Yuri A; Trudell, James R; Benavidez, Jillian M; Betz, Heinrich; Harris, R Adron

    2013-02-01

    Ethanol is a widely used drug, yet an understanding of its sites and mechanisms of action remains incomplete. Among the protein targets of ethanol are glycine receptors (GlyRs), which are potentiated by millimolar concentrations of ethanol. In addition, zinc ions also modulate GlyR function, and recent evidence suggests that physiologic concentrations of zinc enhance ethanol potentiation of GlyRs. Here, we first built a homology model of a zinc-bound GlyR using the D80 position as a coordination site for a zinc ion. Next, we investigated in vitro the effects of zinc on ethanol action at recombinant wild-type (WT) and mutant α1 GlyRs containing the D80A substitution, which eliminates zinc potentiation. At D80A GlyRs, the effects of 50 and 200 mM ethanol were reduced as compared with WT receptors. Also, in contrast to what was seen with WT GlyRs, neither adding nor chelating zinc changed the magnitude of ethanol enhancement of mutant D80A receptors. Next, we evaluated the in vivo effects of the D80A substitution by using heterozygous Glra1(D80A) knock-in (KI) mice. The KI mice showed decreased ethanol consumption and preference, and they displayed increased startle responses compared with their WT littermates. Other behavioral tests, including ethanol-induced motor incoordination and strychnine-induced convulsions, revealed no differences between the KI and WT mice. Together, our findings indicate that zinc is critical in determining the effects of ethanol at GlyRs and suggest that zinc binding at the D80 position may be important for mediating some of the behavioral effects of ethanol action at GlyRs.

  14. Expression of NR1 subunit of NMDA receptor and PSD-93/95 in rat hippocampus affected by NR1/NR2 antisense oligodeoxynucleotide

    Czech Academy of Sciences Publication Activity Database

    Vrajová, M.; Bubeníková-Valešová, V.; Klaschka, Jan

    2010-01-01

    Roč. 9, Suppl.1 (2010), S147 ISSN 1744-859X. [International Congress on Neurobiology and Clinical Psychopharmacology and European Psychiatric Association Conference on Treatment Guidance /1./. 19.11.2009-22.11.2009, Thessaloniki] R&D Projects: GA ČR GD309/09/H072; GA MŠk(CZ) 1M0517 Institutional research plan: CEZ:AV0Z10300504 Keywords : schizophrenia * animal model * NMDA receptor * aODN-NR1/NR2 * PPI Subject RIV: FL - Psychiatry, Sexuology

  15. The neurotensin receptor-1 pathway contributes to human ductal breast cancer progression.

    Science.gov (United States)

    Dupouy, Sandra; Viardot-Foucault, Véronique; Alifano, Marco; Souazé, Frédérique; Plu-Bureau, Geneviève; Chaouat, Marc; Lavaur, Anne; Hugol, Danielle; Gespach, Christian; Gompel, Anne; Forgez, Patricia

    2009-01-01

    The neurotensin (NTS) and its specific high affinity G protein coupled receptor, the NT1 receptor (NTSR1), are considered to be a good candidate for one of the factors implicated in neoplastic progression. In breast cancer cells, functionally expressed NT1 receptor coordinates a series of transforming functions including cellular migration and invasion. we investigated the expression of NTS and NTSR1 in normal human breast tissue and in invasive ductal breast carcinomas (IDCs) by immunohistochemistry and RT-PCR. NTS is expressed and up-regulated by estrogen in normal epithelial breast cells. NTS is also found expressed in the ductal and invasive components of IDCs. The high expression of NTSR1 is associated with the SBR grade, the size of the tumor, and the number of metastatic lymph nodes. Furthermore, the NTSR1 high expression is an independent factor of prognosis associated with the death of patients. these data support the activation of neurotensinergic deleterious pathways in breast cancer progression.

  16. CXC chemokine receptor 2 contributes to host defense in murine urinary tract infection

    NARCIS (Netherlands)

    Olszyna, D. P.; Florquin, S.; Sewnath, M.; Branger, J.; Speelman, P.; van Deventer, S. J.; Strieter, R. M.; van der Poll, T.

    2001-01-01

    CXC chemokines have been implicated in the recruitment of neutrophils to sites of infection. To determine the role of CXC chemokines in the host response to urinary tract infection (UTI), female mice were treated with an antibody against the major CXC chemokine receptor in the mouse, CXCR2, before

  17. Interaction of the p85 subunit of PI 3-kinase and its N-terminal SH2 domain with a PDGF receptor phosphorylation site: structural features and analysis of conformational changes.

    Science.gov (United States)

    Panayotou, G; Bax, B; Gout, I; Federwisch, M; Wroblowski, B; Dhand, R; Fry, M J; Blundell, T L; Wollmer, A; Waterfield, M D

    1992-01-01

    Circular dichroism and fluorescence spectroscopy were used to investigate the structure of the p85 alpha subunit of the PI 3-kinase, a closely related p85 beta protein, and a recombinant SH2 domain-containing fragment of p85 alpha. Significant spectral changes, indicative of a conformational change, were observed on formation of a complex with a 17 residue peptide containing a phosphorylated tyrosine residue. The sequence of this peptide is identical to the sequence surrounding Tyr751 in the kinase-insert region of the platelet-derived growth factor beta-receptor (beta PDGFR). The rotational correlation times measured by fluorescence anisotropy decay indicated that phosphopeptide binding changed the shape of the SH2 domain-containing fragment. The CD and fluorescence spectroscopy data support the secondary structure prediction based on sequence analysis and provide evidence for flexible linker regions between the various domains of the p85 proteins. The significance of these results for SH2 domain-containing proteins is discussed. Images PMID:1330535

  18. A multilevel prediction of physiological response to challenge: Interactions among child maltreatment, neighborhood crime, endothelial nitric oxide synthase gene (eNOS), and GABA(A) receptor subunit alpha-6 gene (GABRA6).

    Science.gov (United States)

    Lynch, Michael; Manly, Jody Todd; Cicchetti, Dante

    2015-11-01

    Physiological response to stress has been linked to a variety of healthy and pathological conditions. The current study conducted a multilevel examination of interactions among environmental toxins (i.e., neighborhood crime and child maltreatment) and specific genetic polymorphisms of the endothelial nitric oxide synthase gene (eNOS) and GABA(A) receptor subunit alpha-6 gene (GABRA6). One hundred eighty-six children were recruited at age 4. The presence or absence of child maltreatment as well as the amount of crime that occurred in their neighborhood during the previous year were determined at that time. At age 9, the children were brought to the lab, where their physiological response to a cognitive challenge (i.e., change in the amplitude of the respiratory sinus arrhythmia) was assessed and DNA samples were collected for subsequent genotyping. The results confirmed that complex Gene × Gene, Environment × Environment, and Gene × Environment interactions were associated with different patterns of respiratory sinus arrhythmia reactivity. The implications for future research and evidence-based intervention are discussed.

  19. Non-hyperfunctioning nodules from multinodular goiters: a minor role in pathogenesis for somatic activating mutations in the TSH-receptor and Gsalpha subunit genes.

    Science.gov (United States)

    Derrien, C; Sonnet, E; Gicquel, I; Le Gall, J Y; Poirier, J Y; David, V; Maugendre, D

    2001-05-01

    Constitutive activation of the cAMP pathway stimulates thyrocyte proliferation. Gain-of-function mutations in Gsalpha protein have already been identified in thyroid nodules which have lost the ability to trap iodine. In contrast, most of the studies failed to detect somatic activating mutations in the thyrotropin receptor (TSH-R) in non-hyperfunctioning thyroid tumors. The aim of this study was to screen for mutations TSH-R exon 10, encoding the whole intracytoplasmic area involved in signal transduction, and Gsalpha exons 8 and 9, containing the two hot-spot codons 201 and 227, in a subset of non-hyperfunctioning nodules from multinodular goiter. Identified by matching ultrasonography and scintiscan, 22 eufunctioning (normal 99Tc uptake) and 15 nonfunctioning (decreased 99Tc uptake) nodules from 27 non-toxic multinodular goiters were isolated. After DNA extraction, TSH-R exon 10 was analyzed by direct sequencing of the PCR products and Gsalpha exons 8 and 9 by Denaturing Gradient Gel Electrophoresis. No mutation of TSH-R or Gsalpha was detected in the 37 nodules analyzed. This absence of mutation, despite the use of two sensitive screening methods associated with the analysis of the TSH-R whole intracytoplasmic area and Gsalpha two hot-spot codons, suggests that TSH-R and Gsalpha play a minor role in the pathogenesis of non-toxic nodules from multinodular goiters.

  20. Contribution of Adsorbed Protein Films to Nanoscopic Vibrations Exhibited by Bacteria Adhering through Ligand-Receptor Bonds.

    Science.gov (United States)

    Song, Lei; Sjollema, Jelmer; Norde, Willem; Busscher, Henk J; van der Mei, Henny C

    2015-09-29

    Bacteria adhering to surfaces exhibit nanoscopic vibrations that depend on the viscoelasticity of the bond. The quantification of the nanoscopic vibrations of bacteria adhering to surfaces provides new opportunities to better understand the properties of the bond through which bacteria adhere and the mechanisms by which they resist detachment. Often, however, bacteria do not adhere to bare surfaces but to adsorbed protein films, on which adhesion involves highly specific ligand-receptor binding next to nonspecific DLVO interaction forces. Here we determine the contribution of adsorbed salivary protein and fibronectin films to vibrations exhibited by adhering streptococci and staphylococci, respectively. The streptococcal strain used has the ability to adhere to adsorbed salivary proteins films through antigen I/II ligand-receptor binding, while the staphylococcal strain used adheres to adsorbed fibronectin films through a proteinaceous ligand-receptor bond. In the absence of ligand-receptor binding, electrostatic interactions had a large impact on vibration amplitudes of adhering bacteria on glass. On an adsorbed salivary protein film, vibration amplitudes of adhering streptococci depended on the film softness as determined by QCM-D and were reduced after film fixation using glutaraldehyde. On a relatively stiff fibronectin film, cross-linking the film in glutaraldehyde hardly reduced its softness, and accordingly fibronectin film softness did not contribute to vibration amplitudes of adhering staphylococci. However, fixation of the staphylococcus-fibronectin bond further decreased vibration amplitudes, while fixation of the streptococcus bond hardly impacted vibration amplitudes. Summarizing, this study shows that both the softness of adsorbed protein films and the properties of the bond between an adhering bacterium and an adsorbed protein film play an important role in bacterial vibration amplitudes. These nanoscopic vibrations reflect the viscoelasticity of the

  1. Glycine and GABAA receptors mediate tonic and phasic inhibitory processes that contribute to prepulse inhibition in the goldfish startle network

    Directory of Open Access Journals (Sweden)

    Paul C.P. Curtin

    2015-03-01

    Full Text Available Prepulse inhibition (PPI is understood as an inhibitory process that attenuates sensory flow during early stages (20-1000ms of information processing. Here, we applied in vivo electrophysiology and pharmacology to determine if prepulse inhibition (PPI is mediated by glycine receptors (GlyRs and/or GABAA receptors (GABAARs in the goldfish auditory startle circuit. Specifically, we used selective antagonists to dissect the contributions of target receptors on sound-evoked postsynaptic potentials (PSPs recorded in the neurons that initiate startle, the Mauthner-cells (M-cell. We found that strychnine, a GlyR antagonist, disrupted a fast-activated (5 ms and rapidly (< 50ms decaying (feed-forward inhibitory process that disrupts PPI at 20 ms prepulse/pulse inter-stimulus intervals (ISI. Additionally we observed increases of the evoked postsynaptic potential (PSP peak amplitude (+87.43 ± 21.53%; N=9 and duration (+204 ± 48.91%, N=9. In contrast, treatment with bicuculline, a GABAAR antagonist, caused a general reduction in PPI across all tested ISIs (20-500 ms, essentially eliminating PPI at ISIs from 20-100 ms. Bicuculline also increased PSP peak amplitude (+133.8 ± 10.3%, N=5 and PSP duration (+284.95 ± 65.64%, N=5. Treatment with either antagonist also tonically increased post-synaptic excitability in the M-cells, reflected by an increase in the magnitude of antidromically-evoked action potentials (APs by 15.07 ± 3.21%, N=7 and 16.23 ± 7.08%, N=5 for strychnine and bicuculline, respectively. These results suggest that GABAARs and GlyRs are functionally segregated to short- and longer-lasting sound-evoked (phasic inhibitory processes that contribute to PPI, with the mediation of tonic inhibition by both receptor systems being critical for gain control within the M-cell startle circuit.

  2. Feeding condition and the relative contribution of different dopamine receptor subtypes to the discriminative stimulus effects of cocaine in rats.

    Science.gov (United States)

    Baladi, Michelle G; Newman, Amy H; France, Charles P

    2014-02-01

    The contribution of dopamine receptor subtypes in mediating the discriminative stimulus effects of cocaine is not fully established. Many drug discrimination studies use food to maintain responding, necessitating food restriction, which can alter drug effects. This study established stimulus control with cocaine (10 mg/kg) in free-feeding and food-restricted rats responding under a schedule of stimulus shock termination (SST) and in food-restricted rats responding under a schedule of food presentation to examine whether feeding condition or the reinforcer used to maintain responding impacts the effects of cocaine. Dopamine receptor agonists and antagonists were examined for their ability to mimic or attenuate, respectively, the effects of cocaine. Apomorphine, quinpirole, and lisuride occasioned >90 % responding on the cocaine-associated lever in free-feeding rats responding under a schedule of SST; apomorphine, but not quinpirole or lisuride, occasioned >90 % responding on the cocaine lever in food-restricted rats responding under a schedule of SST. In food-restricted rats responding for food these drugs occasioned little cocaine lever responding and were comparatively more potent in decreasing responding. In free-feeding rats, the effects of cocaine were attenuated by the D2/D3 receptor antagonist raclopride and the D3 receptor-selective antagonist PG01037. In food-restricted rats, raclopride and the D2 receptor-selective antagonist L-741,626 attenuated the effects of cocaine. Raclopride antagonized quinpirole in all groups while PG01037 antagonized quinpirole only in free-feeding rats. These results demonstrate significant differences in the discriminative stimulus of cocaine that are due to feeding conditions and not to the use of different reinforcers across procedures.

  3. Dopamine D4 receptor stimulation contributes to novel object recognition: Relevance to cognitive impairment in schizophrenia.

    Science.gov (United States)

    Miyauchi, Masanori; Neugebauer, Nichole M; Meltzer, Herbert Y

    2017-04-01

    Several atypical antipsychotic drugs (APDs) have high affinity for the dopamine (DA) D 4 receptor, but the relevance to the efficacy for the treatment of cognitive impairment associated with schizophrenia (CIAS) is poorly understood. The aim of this study was to investigate the effects of D 4 receptor stimulation or blockade on novel object recognition (NOR) in normal rats and on the sub-chronic phencyclidine (PCP)-induced novel object recognition deficit. The effect of the D 4 agonist, PD168077, and the D 4 antagonist, L-745,870, were studied alone, and in combination with clozapine and lurasidone. In normal rats, L-745,870 impaired novel object recognition, whereas PD168077 had no effect. PD168077 acutely reversed the sub-chronic phencyclidine-induced novel object recognition deficit. Co-administration of a sub-effective dose (SED) of PD168077 with a sub-effective dose of lurasidone also reversed this deficit, but a sub-effective dose of PD168077 with a sub-effective dose of clozapine, a more potent D 4 antagonist than lurasidone, did not reverse the sub-chronic phencyclidine-induced novel object recognition deficit. At a dose that did not induce a novel object recognition deficit, L-745,870 blocked the ability of clozapine, but not lurasidone, to reverse the novel object recognition deficit. D 4 receptor agonism has a beneficial effect on novel object recognition in sub-chronic PCP-treated rats and augments the cognitive enhancing efficacy of an atypical antipsychotic drug that lacks affinity for the D 4 receptor, lurasidone.

  4. Activation of NMDA receptor by elevated homocysteine in chronic liver disease contributes to encephalopathy.

    Science.gov (United States)

    Choudhury, Sabanum; Borah, Anupom

    2015-07-01

    Liver diseases lead to a complex syndrome characterized by neurological, neuro-psychiatric and motor complications, called hepatic encephalopathy, which is prevalent in patients and animal models of acute, sub-chronic and chronic liver failure. Although alterations in GABAergic, glutamatergic, cholinergic and serotonergic neuronal functions have been implicated in HE, the molecular mechanisms that lead to HE in chronic liver disease (CLD) is least illustrated. Due to hepatocellular failure, levels of ammonia and homocysteine (Hcy), in addition to others, are found to increase in the brain as well as plasma. Hcy, a non-protein forming amino acid and an excitotoxin, activates ionotropic glutamate (n-methyl-d-aspartate; NMDA) receptors, and thereby leads to influx of Ca(2+) into neurons, which in turn activates several pathways that trigger oxidative stress, inflammation and apoptosis, collectively called excitotoxicity. Elevated levels of Hcy in the plasma and brain, a condition called Hyperhomocysteinemia (HHcy), and the resultant NMDA receptor-mediated excitotoxicity has been implicated in several diseases, including Parkinson's disease and Alzheimer's disease. Although, hyperammonemia has been shown to cause excitotoxicity, the role of HHcy in the development of behavioral and neurochemical alterations that occur in HE has not been illustrated yet. It is hypothesized that CLD-induced HHcy plays a major role in the development of HE through activation of NMDA receptors. It is further hypothesized that HHcy synergizes with hyperammonemia to activate NMDA receptor in the brain, and thereby cause oxidative stress, inflammation and apoptosis, and neuronal loss that leads to HE. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Somatic hypermutation of T cell receptor α chain contributes to selection in nurse shark thymus.

    Science.gov (United States)

    Ott, Jeannine A; Castro, Caitlin D; Deiss, Thaddeus C; Ohta, Yuko; Flajnik, Martin F; Criscitiello, Michael F

    2018-04-17

    Since the discovery of the T cell receptor (TcR), immunologists have assigned somatic hypermutation (SHM) as a mechanism employed solely by B cells to diversify their antigen receptors. Remarkably, we found SHM acting in the thymus on α chain locus of shark TcR. SHM in developing shark T cells likely is catalyzed by activation-induced cytidine deaminase (AID) and results in both point and tandem mutations that accumulate non-conservative amino acid replacements within complementarity-determining regions (CDRs). Mutation frequency at TcRα was as high as that seen at B cell receptor loci (BcR) in sharks and mammals, and the mechanism of SHM shares unique characteristics first detected at shark BcR loci. Additionally, fluorescence in situ hybridization showed the strongest AID expression in thymic corticomedullary junction and medulla. We suggest that TcRα utilizes SHM to broaden diversification of the primary αβ T cell repertoire in sharks, the first reported use in vertebrates. © 2018, Ott et al.

  6. The neurotensin receptor-1 pathway contributes to human ductal breast cancer progression.

    Directory of Open Access Journals (Sweden)

    Sandra Dupouy

    Full Text Available BACKGROUND: The neurotensin (NTS and its specific high affinity G protein coupled receptor, the NT1 receptor (NTSR1, are considered to be a good candidate for one of the factors implicated in neoplastic progression. In breast cancer cells, functionally expressed NT1 receptor coordinates a series of transforming functions including cellular migration and invasion. METHODS AND RESULTS: we investigated the expression of NTS and NTSR1 in normal human breast tissue and in invasive ductal breast carcinomas (IDCs by immunohistochemistry and RT-PCR. NTS is expressed and up-regulated by estrogen in normal epithelial breast cells. NTS is also found expressed in the ductal and invasive components of IDCs. The high expression of NTSR1 is associated with the SBR grade, the size of the tumor, and the number of metastatic lymph nodes. Furthermore, the NTSR1 high expression is an independent factor of prognosis associated with the death of patients. CONCLUSION: these data support the activation of neurotensinergic deleterious pathways in breast cancer progression.

  7. Dissociable Hippocampal and Amygdalar D1-like receptor contribution to Discriminated Pavlovian conditioned approach learning

    Science.gov (United States)

    Andrzejewski, Matthew E; Ryals, Curtis

    2016-01-01

    Pavlovian conditioning is an elementary form of reward-related behavioral adaptation. The mesolimbic dopamine system is widely considered to mediate critical aspects of reward-related learning. For example, initial acquisition of positively-reinforced operant behavior requires dopamine (DA) D1 receptor (D1R) activation in the basolateral amygdala (BLA), central nucleus of the amygdala (CeA), and the ventral subiculum (vSUB). However, the role of D1R activation in these areas on appetitive, non-drug-related, Pavlovian learning is not currently known. In separate experiments, microinfusions of the D1-like receptor antagonist SCH-23390 (3.0 nmol/0.5 μL per side) into the amygdala and subiculum preceded discriminated Pavlovian conditioned approach (dPCA) training sessions. D1-like antagonism in all three structures impaired the acquisition of discriminated approach, but had no effect on performance after conditioning was asymptotic. Moreover, dissociable effects of D1-like antagonism in the three structures on components of discriminated responding were obtained. Lastly, the lack of latent inhibition in drug-treated groups may elucidate the role of D1-like in reward-related Pavlovian conditioning. The present data suggest a role for the D1 receptors in the amygdala and hippocampus in learning the significance of conditional stimuli, but not in the expression of conditional responses. PMID:26632336

  8. NMDA receptor function during senescence: implication on cognitive performance

    Directory of Open Access Journals (Sweden)

    Ashok eKumar

    2015-12-01

    Full Text Available N-methyl-D-aspartate (NMDA receptors, a family of L-glutamate receptors, play an important role in learning and memory, and are critical for spatial memory. These receptors are tetrameric ion channels composed of a family of related subunits. One of the hallmarks of the aging human population is a decline in cognitive function; studies in the past couple of years have demonstrated deterioration in NMDA receptor subunit expression and function with advancing age. However, a direct relationship between impaired memory function and a decline in NMDA receptors is still ambiguous. Recent studies indicate a link between an age-associated NMDA receptor hypofunction and memory impairment and provide evidence that age-associated enhanced oxidative stress might be contributing to the alterations associated with senescence. However, clear evidence is still deficient in demonstrating the underlying mechanisms and a relationship between age-associated impaired cognitive faculties and NMDA receptor hypofunction. The current review intends to present an overview of the research findings regarding changes in expression of various NMDA receptor subunits and deficits in NMDA receptor function during senescence and its implication in age-associated impaired hippocampal-dependent memory function.

  9. Peripheral and central P2X3 receptor contributions to colon mechanosensitivity and hypersensitivity in the mouse

    Science.gov (United States)

    Shinoda, Masamichi; Feng, Bin; Gebhart, G. F.

    2009-01-01

    Background & Aims Irritable bowel syndrome is characterized by altered sensory qualities, namely discomfort/pain and colorectal hypersensitivity. In mice, we examined the role of P2X3 receptors in colon mechanosensitivity and intracolonic zymosan-produced hypersensitivity, a model of persistent colon hypersensitivity without colon inflammation. Methods The visceromotor response (VMR) to colon distension (15 – 60 mmHg) was determined before and after intracolonic saline or zymosan (30 mg/mL, 0.1 mL, daily for 3 days) treatment. Colon pathology and intracolonic ATP release was assessed in parallel experiments. To examine P2X3 receptor contributions to colon mechanosensation and hypersensitivity, electrophysiological experiments were performed using an in vitro colon-pelvic nerve preparation. Results VMRs to distension were significantly reduced in P2X3+/−and P2X3−/− mice relative to wildtype mice. Colon hypersensitivity produced by zymosan was virtually absent in P2X3−/− relative to wildtype or P2X3+/− mice. Intralumenal release of the endogenous P2X receptor ligand ATP did not differ between wildtype and P2X3−/− mice or change after intracolonic zymosan treatment. Responses of muscular and muscular-mucosal pelvic nerve afferents to mechanical stretch did not differ between P2X3−/− and wildtype mice. Both muscular and muscular-mucosal afferents in wildtype mice sensitized to application of an inflammatory soup, whereas only muscular-mucosal afferents did so in P2X3−/− mice. Conclusions These results suggest differential roles for peripheral and central P2X3 receptors in colon mechanosensory transduction and hypersensitivity. PMID:19549524

  10. Upregulation of the sodium channel NaVβ4 subunit and its contributions to mechanical hypersensitivity and neuronal hyperexcitability in a rat model of radicular pain induced by local DRG inflammation

    Science.gov (United States)

    Xie, Wenrui; Tan, Zhi-Yong; Barbosa, Cindy; Strong, Judith A.; Cummins, Theodore R.; Zhang, Jun-Ming

    2016-01-01

    High frequency spontaneous firing in myelinated sensory neurons plays a key role in initiating pain behaviors in several different models, including the radicular pain model in which the rat lumbar dorsal root ganglia (DRG) are locally inflamed. The sodium channel isoform NaV1.6 contributes to pain behaviors and spontaneous activity in this model. Among all the isoforms in adult DRG, NaV1.6 is the main carrier of TTX-sensitive resurgent Na currents that allow high-frequency firing. Resurgent currents flow after a depolarization or action potential, as a blocking particle exits the pore. In most neurons the regulatory β4 subunit is potentially the endogenous blocker. We used in vivo siRNA mediated knockdown of NaVβ4 to examine its role in the DRG inflammation model. NaVβ4 but not control siRNA almost completely blocked mechanical hypersensitivity induced by DRG inflammation. Microelectrode recordings in isolated whole DRGs showed that NaVβ4 siRNA blocked the inflammation-induced increase in spontaneous activity of Aβ neurons, and reduced repetitive firing and other measures of excitability. NaVβ4 was preferentially expressed in larger diameter cells; DRG inflammation increased its expression and this was reversed by NaVβ4 siRNA, based on immunohistochemistry and Western blotting. NaVβ4 siRNA also reduced immunohistochemical NaV1.6 expression. Patch clamp recordings of TTX-sensitive Na currents in acutely cultured medium diameter DRG neurons showed that DRG inflammation increased transient and especially resurgent current; effects blocked by NaVβ4 siRNA. NaVβ4 may represent a more specific target for pain conditions that depend on myelinated neurons expressing NaV1.6. PMID:26785322

  11. Inverse agonism at the P2Y12 receptor and ENT1 transporter blockade contribute to platelet inhibition by ticagrelor.

    Science.gov (United States)

    Aungraheeta, Riyaad; Conibear, Alexandra; Butler, Mark; Kelly, Eamonn; Nylander, Sven; Mumford, Andrew; Mundell, Stuart J

    2016-12-08

    Ticagrelor is a potent antagonist of the P2Y 12 receptor (P2Y 12 R) and consequently an inhibitor of platelet activity effective in the treatment of atherothrombosis. Here, we sought to further characterize its molecular mechanism of action. Initial studies showed that ticagrelor promoted a greater inhibition of adenosine 5'-diphosphate (ADP)-induced Ca 2+ release in washed platelets vs other P2Y 12 R antagonists. This additional effect of ticagrelor beyond P2Y 12 R antagonism was in part as a consequence of ticagrelor inhibiting the equilibrative nucleoside transporter 1 (ENT1) on platelets, leading to accumulation of extracellular adenosine and activation of G s -coupled adenosine A 2A receptors. This contributed to an increase in basal cyclic adenosine monophosphate (cAMP) and vasodilator-stimulated phosphoprotein phosphorylation (VASP-P). In addition, ticagrelor increased platelet cAMP and VASP-P in the absence of ADP in an adenosine receptor-independent manner. We hypothesized that this increase originated from a direct effect on basal agonist-independent P2Y 12 R signaling, and this was validated in 1321N1 cells stably transfected with human P2Y 12 R. In these cells, ticagrelor blocked the constitutive agonist-independent activity of the P2Y 12 R, limiting basal G i -coupled signaling and thereby increasing cAMP levels. These data suggest that ticagrelor has the pharmacological profile of an inverse agonist. Based on our results showing insurmountable inhibition of ADP-induced Ca 2+ release and forskolin-induced cAMP, the mode of antagonism of ticagrelor also appears noncompetitive, at least functionally. In summary, our studies describe 2 novel modes of action of ticagrelor, inhibition of platelet ENT1 and inverse agonism at the P2Y 12 R that contribute to its effective inhibition of platelet activation. © 2016 by The American Society of Hematology.

  12. Autophagy downregulation contributes to insulin resistance mediated injury in insulin receptor knockout podocytes in vitro

    Directory of Open Access Journals (Sweden)

    Ying Xu

    2016-04-01

    Full Text Available It is unknown whether autophagy activity is altered in insulin resistant podocytes and whether autophagy could be a therapeutic target for diabetic nephropathy (DN. Here we used shRNA transfection to knockdown the insulin receptor (IR gene in cultured human immortalized podocytes as an in vitro insulin resistant model. Autophagy related proteins LC3, Beclin, and p62 as well as nephrin, a podocyte injury marker, were assessed using western blot and immunofluorescence staining. Our results show that autophagy is suppressed when podocytes lose insulin sensitivity and that treatment of rapamycin, an mTOR specific inhibitor, could attenuate insulin resistance induced podocytes injury via autophagy activation. The present study deepens our understanding of the role of autophagy in the pathogenesis of DN.

  13. The receptor for advanced glycation end products (RAGE contributes to the progression of emphysema in mice.

    Directory of Open Access Journals (Sweden)

    Nisha Sambamurthy

    Full Text Available Several recent clinical studies have implied a role for the receptor for advanced glycation end products (RAGE and its variants in chronic obstructive pulmonary disease (COPD. In this study we have defined a role for RAGE in the pathogenesis of emphysema in mice. RAGE deficient mice (RAGE-/- exposed to chronic cigarette smoke were significantly protected from smoke induced emphysema as determined by airspace enlargement and had no significant reduction in lung tissue elastance when compared to their air exposed controls contrary to their wild type littermates. The progression of emphysema has been largely attributed to an increased inflammatory cell-mediated elastolysis. Acute cigarette smoke exposure in RAGE-/- mice revealed an impaired early recruitment of neutrophils, approximately a 6-fold decrease compared to wild type mice. Hence, impaired neutrophil recruitment with continued cigarette smoke exposure reduces elastolysis and consequent emphysema.

  14. The gram-negative sensing receptor PGRP-LC contributes to grooming induction in Drosophila.

    Science.gov (United States)

    Yanagawa, Aya; Neyen, Claudine; Lemaitre, Bruno; Marion-Poll, Frédéric

    2017-01-01

    Behavioral resistance protects insects from microbial infection. However, signals inducing insect hygiene behavior are still relatively unexplored. Our previous study demonstrated that olfactory signals from microbes enhance insect hygiene behavior, and gustatory signals even induce the behavior. In this paper, we postulated a cross-talk between behavioral resistance and innate immunity. To examine this hypothesis, we employed a previously validated behavioral test to examine the function of taste signals in inducing a grooming reflex in decapitated flies. Microbes, which activate different pattern recognition systems upstream of immune pathways, were applied to see if there was any correlation between microbial perception and grooming reflex. To narrow down candidate elicitors, the grooming induction tests were conducted with highly purified bacterial components. Lastly, the role of DAP-type peptidoglycan in grooming induction was confirmed. Our results demonstrate that cleaning behavior can be triggered through recognition of DAP-type PGN by its receptor PGRP-LC.

  15. Adenosine A1 receptors contribute to immune regulation after neonatal hypoxic ischemic brain injury.

    Science.gov (United States)

    Winerdal, Max; Winerdal, Malin E; Wang, Ying-Qing; Fredholm, Bertil B; Winqvist, Ola; Ådén, Ulrika

    2016-03-01

    Neonatal brain hypoxic ischemia (HI) often results in long-term motor and cognitive impairments. Post-ischemic inflammation greatly effects outcome and adenosine receptor signaling modulates both HI and immune cell function. Here, we investigated the influence of adenosine A1 receptor deficiency (A1R(-/-)) on key immune cell populations in a neonatal brain HI model. Ten-day-old mice were subjected to HI. Functional outcome was assessed by open locomotion and beam walking test and infarction size evaluated. Flow cytometry was performed on brain-infiltrating cells, and semi-automated analysis of flow cytometric data was applied. A1R(-/-) mice displayed larger infarctions (+33%, p beam walking tests (44% more mistakes, p < 0.05) than wild-type (WT) mice. Myeloid cell activation after injury was enhanced in A1R(-/-) versus WT brains. Activated B lymphocytes expressing IL-10 infiltrated the brain after HI in WT, but were less activated and did not increase in relative frequency in A1R(-/-). Also, A1R(-/-) B lymphocytes expressed less IL-10 than their WT counterparts, the A1R antagonist DPCPX decreased IL-10 expression whereas the A1R agonist CPA increased it. CD4(+) T lymphocytes including FoxP3(+) T regulatory cells, were unaffected by genotype, whereas CD8(+) T lymphocyte responses were smaller in A1R(-/-) mice. Using PCA to characterize the immune profile, we could discriminate the A1R(-/-) and WT genotypes as well as sham operated from HI-subjected animals. We conclude that A1R signaling modulates IL-10 expression by immune cells, influences the activation of these cells in vivo, and affects outcome after HI.

  16. Contribution of glucocorticoids and glucocorticoid receptors to the regulation of neurodegenerative processes.

    Science.gov (United States)

    Vyas, Sheela; Maatouk, Layal

    2013-12-01

    Isolation of glucocorticoids (GCs) from adrenal glands followed by synthesis led rapidly to their first clinical application, about 70 years ago, for treatment of rheumatoid arthritis. To this day GCs are used in diseases that have an inflammatory component. However, their use is carefully monitored because of harmful side effects. GCs are also synonymous with stress and adaptation. In CNS, GC binds and activates high affinity mineralocorticoid receptor (MR) and low affinity glucocorticoid receptor (GR). GR, whose expression is ubiquitous, is only activated when GC levels rise as during circadian peak and in response to stress. Numerous recent studies have yielded important and new insights on the mechanisms concerning pulsatile secretory pattern of GCs as well as various processes that tightly control their synthesis via hypothalamic-pituitary-adrenal (HPA) axis involving regulated release of corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH) from hypothalamus and pituitary, respectively. GR modulates neuronal functions and viability through both genomic and non-genomic actions, and importantly its transcriptional regulatory activity is tightly locked with GC secretory pattern. There is increasing evidence pointing to involvement of GC-GR in neurodegenerative disorders. Patients with Alzheimer's or Parkinson's or Huntington's disease show chronically high cortisol levels suggesting changes occurring in controls of HPA axis. In experimental models of these diseases, chronic stress or GC treatment was found to exacerbate both the clinical symptoms and neurodegenerative processes. However, recent evidence also shows that GC-GR can exert neuroprotective effects. Thus, for any potential therapeutic strategies in these neurodegenerative diseases we need to understand the precise modifications both in HPA axis and in GR activity and find ways to harness their protective actions.

  17. Dissociation between neural and vascular responses to sympathetic stimulation : contribution of local adrenergic receptor function

    Science.gov (United States)

    Jacob, G.; Costa, F.; Shannon, J.; Robertson, D.; Biaggioni, I.

    2000-01-01

    Sympathetic activation produced by various stimuli, eg, mental stress or handgrip, evokes regional vascular responses that are often nonhomogeneous. This phenomenon is believed to be the consequence of the recruitment of differential central neural pathways or of a sympathetically mediated vasodilation. The purpose of this study was to determine whether a similar heterogeneous response occurs with cold pressor stimulation and to test the hypothesis that local differences in adrenergic receptor function could be in part responsible for this diversity. In 8 healthy subjects, local norepinephrine spillover and blood flow were measured in arms and legs at baseline and during sympathetic stimulation induced by baroreflex mechanisms (nitroprusside infusion) or cold pressor stimulation. At baseline, legs had higher vascular resistance (27+/-5 versus 17+/-2 U, P=0.05) despite lower norepinephrine spillover (0.28+/-0.04 versus 0.4+/-0.05 mg. min(-1). dL(-1), P=0.03). Norepinephrine spillover increased similarly in both arms and legs during nitroprusside infusion and cold pressor stimulation. On the other hand, during cold stimulation, vascular resistance increased in arms but not in legs (20+/-9% versus -7+/-4%, P=0.03). Increasing doses of isoproterenol and phenylephrine were infused intra-arterially in arms and legs to estimate beta-mediated vasodilation and alpha-induced vasoconstriction, respectively. beta-Mediated vasodilation was significantly lower in legs compared with arms. Thus, we report a dissociation between norepinephrine spillover and vascular responses to cold stress in lower limbs characterized by a paradoxical decrease in local resistance despite increases in sympathetic activity. The differences observed in adrenergic receptor responses cannot explain this phenomenon.

  18. Epilepsy, E/I balance and GABAA receptor plasticity

    Directory of Open Access Journals (Sweden)

    Jean-Marc Fritschy

    2008-03-01

    Full Text Available GABAA receptors mediate most of the fast inhibitory transmission in the CNS. They form heteromeric complexes assembled from a large family of subunit genes. The existence of multiple GABAA receptor subtypes differing in subunit composition, localization and functional properties underlies their role for fi ne-tuning of neuronal circuits and genesis of network oscillations. The differential regulation of GABAA receptor subtypes represents a major facet of homeostatic synaptic plasticity and contributes to the excitation/inhibition (E/I balance under physiological conditions and upon pathological challenges. The purpose of this review is to discuss recent fi ndings highlighting the signifi cance of GABAA receptor heterogeneity for the concept of E/I balance and its relevance for epilepsy. Specifi cally, we address the following issues: (1 role for tonic inhibition, mediated by extrasynaptic GABAA receptors, for controlling neuronal excitability; (2 signifi cance of chloride ion transport for maintenance of the E/I balance in adult brain; and (3 molecular mechanisms underlying GABAA receptor regulation (traffi cking, posttranslational modifi cation, gene transcription that are important for homoeostatic plasticity. Finally, the relevance of these fi ndings is discussed in light of the involvement of GABAA receptors in epileptic disorders, based on recent experimental studies of temporal lobe epilepsy (TLE and absence seizures and on the identifi cation of mutations in GABAA receptor subunit genes underlying familial forms of epilepsy.

  19. Enhanced long-term and impaired short-term spatial memory in GluA1 AMPA receptor subunit knockout mice: evidence for a dual-process memory model.

    Science.gov (United States)

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

    2009-06-01

    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 GluA1-dependent synaptic plasticity for short-term memory of recently visited places, but not for the ability to form long-term associations between a particular spatial location and an outcome. This hypothesis is in concordance with the theory that short-term and long-term memory depend on dissociable psychological processes. In this study we tested GluA1-/- mice on both short-term and long-term spatial memory using a simple novelty preference task. Mice were given a series of repeated exposures to a particular spatial location (the arm of a Y-maze) before their preference for a novel spatial location (the unvisited arm of the maze) over the familiar spatial location was assessed. GluA1-/- mice were impaired if the interval between the trials was short (1 min), but showed enhanced spatial memory if the interval between the trials was long (24 h). This enhancement was caused by the interval between the exposure trials rather than the interval prior to the test, thus demonstrating enhanced learning and not simply enhanced performance or expression of memory. This seemingly paradoxical enhancement of hippocampus-dependent spatial learning may be caused by GluA1 gene deletion reducing the detrimental effects of short-term memory on subsequent long-term learning. Thus, these results support a dual-process model of memory in which short-term and long-term memory are separate and sometimes competitive processes.

  20. Repeated intermittent alcohol exposure during the third trimester-equivalent increases expression of the GABA(A) receptor δ subunit in cerebellar granule neurons and delays motor development in rats.

    Science.gov (United States)

    Diaz, Marvin R; Vollmer, Cyndel C; Zamudio-Bulcock, Paula A; Vollmer, William; Blomquist, Samantha L; Morton, Russell A; Everett, Julie C; Zurek, Agnieszka A; Yu, Jieying; Orser, Beverley A; Valenzuela, C Fernando

    2014-04-01

    Exposure to ethanol (EtOH) during fetal development can lead to long-lasting alterations, including deficits in fine motor skills and motor learning. Studies suggest that these are, in part, a consequence of cerebellar damage. Cerebellar granule neurons (CGNs) are the gateway of information into the cerebellar cortex. Functionally, CGNs are heavily regulated by phasic and tonic GABAergic inhibition from Golgi cell interneurons; however, the effect of EtOH exposure on the development of GABAergic transmission in immature CGNs has not been investigated. To model EtOH exposure during the 3rd trimester-equivalent of human pregnancy, neonatal pups were exposed intermittently to high levels of vaporized EtOH from postnatal day (P) 2 to P12. This exposure gradually increased pup serum EtOH concentrations (SECs) to ∼60 mM (∼0.28 g/dl) during the 4 h of exposure. EtOH levels gradually decreased to baseline 8 h after the end of exposure. Surprisingly, basal tonic and phasic GABAergic currents in CGNs were not significantly affected by postnatal alcohol exposure (PAE). However, PAE increased δ subunit expression at P28 as detected by immunohistochemical and western blot analyses. Also, electrophysiological studies with an agonist that is highly selective for δ-containing GABA(A) receptors, 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridine-3-ol (THIP), showed an increase in THIP-induced tonic current. Behavioral studies of PAE rats did not reveal any deficits in motor coordination, except for a delay in the acquisition of the mid-air righting reflex that was apparent at P15 to P18. These findings demonstrate that repeated intermittent exposure to high levels of EtOH during the equivalent of the last trimester of human pregnancy has significant but relatively subtle effects on motor coordination and GABAergic transmission in CGNs in rats. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. Zebrafish Adar2 Edits the Q/R site of AMPA receptor Subunit gria2α transcript to ensure normal development of nervous system and cranial neural crest cells.

    Directory of Open Access Journals (Sweden)

    I-Chen Li

    Full Text Available BACKGROUND: Adar2 deaminates selective adenosines to inosines (A-to-I RNA editing in the double-stranded region of nuclear transcripts. Although the functions of mouse Adar2 and its biologically most important substrate gria2, encoding the GluA2 subunit of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor, have been extensively studied, the substrates and functions of zebrafish Adar2 remain elusive. METHODS/PRINCIPAL FINDINGS: Expression of Adar2 was perturbed in the adar2 morphant (adar2MO, generated by antisense morpholio oligonucleotides. The Q/R editing of gria2α was reduced in the adar2MO and was enhanced by overexpression of Adar2, demonstrating an evolutionarily conserved activity between zebrafish and mammalian Adar2 in editing the Q/R site of gria2. To delineate the role of Q/R editing of gria2α in the developmental defects observed in the adar2MO, the Q/R editing of gria2α was specifically perturbed in the gria2αQRMO, generated by a morpholio oligonucleotide complementary to the exon complementary sequence (ECS required for the Q/R editing. Analogous to the adar2-deficient and Q/R-editing deficient mice displaying identical neurological defects, the gria2αQRMO and adar2MO displayed identical developmental defects in the nervous system and cranial cartilages. Knockdown p53 abolished apoptosis and partially suppressed the loss of spinal cord motor neurons in these morphants. However, reducing p53 activity neither replenished the brain neuronal populations nor rescued the developmental defects. The expressions of crestin and sox9b in the neural crest cells were reduced in the adar2MO and gria2αQRMO. Overexpressing the edited GluA2αR in the adar2MO restored normal expressions of cresting and sox9b. Moreover, overexpressing the unedited GluA2αQ in the wild type embryos resulted in reduction of crestin and sox9b expressions. These results argue that an elevated GluA2αQ level is sufficient for generating the

  2. Upregulation of vascular endothelial growth factor receptor-1 contributes to sevoflurane preconditioning–mediated cardioprotection

    Directory of Open Access Journals (Sweden)

    Qian B

    2018-04-01

    Full Text Available Bin Qian,1 Yang Yang,2 Yusheng Yao,3 Yanling Liao,3 Ying Lin3 1Department of Anesthesiology, People’s Hospital Affiliated to Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian, China; 2Department of Anesthesiology, West China Hospital, Sichuan University, Chengdu, Sichuan, China; 3Department of Anesthesiology, The Shengli Clinical Medical College, Fujian Medical University, Fuzhou, Fujian, China Purpose: Sevoflurane preconditioning (SPC can provide myocardial protective effects similar to ischemic preconditioning. However, the exact mechanism of SPC remains unclear. Previous studies indicate that vascular endothelial growth factor receptor 1 (VEGFR-1 is involved in ischemic preconditioning-mediated cardioprotection. This study was designed to determine the significance of VEGFR-1 signaling in SPC-mediated cardioprotection.Materials and methods: Myocardial ischemia–reperfusion (I/R rat model was established using the Langendorff isolated heart perfusion apparatus. Additionally, after 15 min of baseline equilibration, the isolated hearts were pretreated with 2.5% sevoflurane, 2.5% sevoflurane+MF1 10 µmol/L, or 2.5% sevoflurane+placental growth factor 10 µmol/L, and then subjected to 30 min of global ischemia and 120 min of reperfusion. The changes in hemodynamic parameters, myocardial infarct size, and the levels of creatine kinase-MB, lactate dehydrogenase, cardiac troponin-I, tumor necrosis factor-α, and interleukin 6 in the myocardium were evaluated.Results: Compared to the I/R group, pretreatment with 2.5% sevoflurane significantly improved the cardiac function, limited myocardial infarct size, reduced cardiac enzyme release, upregulated VEGFR-1 expression, and decreased inflammation. In addition, the selective VEGFR-1 agonist, placental growth factor, did not enhance the cardioprotection and anti-inflammation effects of sevoflurane, while the specific VEGFR-1 inhibitor, MF1, completely reversed these effects

  3. Depression in early adolescence: Contributions from relational aggression and variation in the oxytocin receptor gene.

    Science.gov (United States)

    Kushner, Shauna C; Herzhoff, Kathrin; Vrshek-Schallhorn, Suzanne; Tackett, Jennifer L

    2018-01-01

    Interpersonal stress arising from relational aggression (RA)-the intentional effort to harm others via rejection and exclusion-may increase risk for depression in youth. Biological vulnerabilities related to the hormone oxytocin, which affects social behavior and stress responses, may exacerbate this risk. In a community sample of 307 youth (52% female; age range = 10-14 years), we tested whether (1) the association between RA and subsequent depressive symptoms was mediated through social problems and (2) a single nucleotide polymorphism (rs53576) in the oxytocin receptor gene (OXTR) moderated this indirect association between RA and depression, where GG homozygotes are predicted to be more sensitive to the effects of social problems than A-allele carriers. Youth-reported RA and depressive symptoms were measured using a structured interview and a questionnaire, respectively. DNA was extracted from saliva collected with Oragene kits. Consistent with the interpersonal theory of depression, the association between relational aggression and subsequent depressive symptoms was mediated by social problems. This indirect effect was further moderated by rs53576 genotype, such that GG homozygotes showed a stronger mediation effect than A-carriers. These results suggest that rs53576 variants confer vulnerability for depression within the context of interpersonal risk factors, such that youth with the GG genotype may be particularly sensitive to the social consequences resulting from RA. © 2017 Wiley Periodicals, Inc.

  4. Transient receptor potential A1 channel contributes to activation of the muscle reflex.

    Science.gov (United States)

    Koba, Satoshi; Hayes, Shawn G; Sinoway, Lawrence I

    2011-01-01

    This study was undertaken to elucidate the role played by transient receptor potential A1 channels (TRPA1) in activating the muscle reflex, a sympathoexcitatory drive originating in contracting muscle. First, we tested the hypothesis that stimulation of the TRPA1 located on muscle afferents reflexly increases sympathetic nerve activity. In decerebrate rats, allyl isothiocyanate, a TRPA1 agonist, was injected intra-arterially into the hindlimb muscle circulation. This led to a 33% increase in renal sympathetic nerve activity (RSNA). The effect of allyl isothiocyanate was a reflex because the response was prevented by sectioning the sciatic nerve. Second, we tested the hypothesis that blockade of TRPA1 reduces RSNA response to contraction. Thirty-second continuous static contraction of the hindlimb muscles, induced by electrical stimulation of the peripheral cut ends of L(4) and L(5) ventral roots, increased RSNA and blood pressure. The integrated RSNA during contraction was reduced by HC-030031, a TRPA1 antagonist, injected intra-arterially (163 ± 24 vs. 95 ± 21 arbitrary units, before vs. after HC-030031, P reflex. Increases in RSNA in response to injection into the muscle circulation of arachidonic acid, bradykinin, and diprotonated phosphate, which are metabolic by-products of contraction and stimulants of muscle afferents during contraction, were reduced by HC-030031. These observations suggest that the TRPA1 located on muscle afferents is part of the muscle reflex and further support the notion that arachidonic acid metabolites, bradykinin, and diprotonated phosphate are candidates for endogenous agonists of TRPA1.

  5. Orphan nuclear receptor TLX contributes to androgen insensitivity in castration-resistant prostate cancer via its repression of androgen receptor transcription.

    Science.gov (United States)

    Jia, Lin; Wu, Dinglan; Wang, Yuliang; You, Wenxing; Wang, Zhu; Xiao, Lijia; Cai, Ganhui; Xu, Zhenyu; Zou, Chang; Wang, Fei; Teoh, Jeremy Yuen-Chun; Ng, Chi-Fai; Yu, Shan; Chan, Franky L

    2018-03-20

    The metastatic castration-resistant prostate cancer (CRPC) is a lethal form of prostate cancer, in which the expression of androgen receptor (AR) is highly heterogeneous. Indeed, lower AR expression and attenuated AR signature activity is shown in CRPC tissues, especially in the subset of neuroendocrine prostate cancer (NEPC) and prostate cancer stem-like cells (PCSCs). However, the significance of AR downregulation in androgen insensitivity and de-differentiation of tumor cells in CRPC is poorly understood and much neglected. Our previous study shows that the orphan nuclear receptor TLX (NR2E1), which is upregulated in prostate cancer, plays an oncogenic role in prostate carcinogenesis by suppressing oncogene-induced senescence. In the present study, we further established that TLX exhibited an increased expression in metastatic CRPC. Further analyses showed that overexpression of TLX could confer resistance to androgen deprivation and anti-androgen in androgen-dependent prostate cancer cells in vitro and in vivo, whereas knockdown of endogenous TLX could potentiate the sensitivity to androgen deprivation and anti-androgen in prostate cancer cells. Our study revealed that the TLX-induced resistance to androgen deprivation and anti-androgen was mediated through its direct suppression of AR gene transcription and signaling in both androgen-stimulated and -unstimulated prostate cancer cells. We also characterized that TLX could bind directly to AR promoter and repress AR transcription by recruitment of histone modifiers, including HDAC1, HDAC3, and LSD1. Together, our present study shows, for the first time, that TLX can contribute to androgen insensitivity in CRPC via repression of AR gene transcription and signaling, and also implicates that targeting the druggable TLX may have a potential therapeutic significance in CRPC management, particularly in NEPC and PCSCs.

  6. Contribution of ventral tegmental GABA receptors to cocaine self-administration in rats.

    Science.gov (United States)

    Backes, E N; Hemby, S E

    2008-03-01

    Recent evidence has suggested that compounds affecting GABAergic transmission may provide useful pharmacological tools for the treatment of cocaine addiction. Using a rat model of self-administration, the present study examined the effects of GABA agonists and antagonists injected directly into the ventral tegmental area (VTA) on cocaine intake in rats trained to self-administer cocaine (0, 125, 250 and 500 microg/infusion) under an FR5 schedule of reinforcement. Separate groups of rats received bilateral intra-VTA injections of the GABA-A antagonist picrotoxin (34 ng/side, n = 7; 68 ng/side, n = 8), GABA-A agonist muscimol (14 ng/side, n = 8), GABA-B agonist baclofen (56 ng/side, n = 7; 100 ng/side, n = 6), picrotoxin (68 ng/side) co-injected with the GABA-B antagonist 2-hydroxysaclofen (100 ng/side, n = 7; 2 microg/side, n = 8) or artificial cerebrospinal fluid (aCSF, n = 6) to assess the effects of the various compounds on the cocaine self-administration dose-response curve. Both picrotoxin and baclofen reduced responding maintained by cocaine, whereas muscimol had no effect on responding. In contrast, neither picrotoxin (n = 6) nor baclofen (n = 8) affected responding maintained by food. Interestingly, 2-hydroxysaclofen effectively blocked the suppression of responding produced by picrotoxin, suggesting that both picrotoxin and baclofen exert their effects via activation of GABA-B receptors. Additionally, these effects appear to be specific to cocaine reinforcement, supporting current investigation of baclofen as a treatment for cocaine addiction.

  7. H pylori receptor MHC class II contributes to the dynamic gastric epithelial apoptotic response

    Science.gov (United States)

    Bland, David A; Suarez, Giovanni; Beswick, Ellen J; Sierra, Johanna C; Reyes, Victor E

    2006-01-01

    AIM: To investigate the role of MHC class II in the modulation of gastric epithelial cell apoptosis induced by H pylori infection. METHODS: After stimulating a human gastric epithelial cell line with bacteria or agonist antibodies specific for MHC class II and CD95, the quantitation of apoptotic and anti-apoptotic events, including caspase activation, BCL-2 activation, and FADD recruitment, was performed with a fluorometric assay, a cytometric bead array, and confocal microscopy, respectively. RESULTS: Pretreatment of N87 cells with the anti-MHC class II IgM antibody RFD1 resulted in a reduction in global caspase activation at 24 h of H pylori infection. When caspase 3 activation was specifically measured, crosslinking of MHC class II resulted in a marked reduced caspase activation, while simple ligation of MHC class II did not. Crosslinking of MHC class II also resulted in an increased activation of the anti-apoptosis molecule BCL-2 compared to simple ligation. Confocal microscope analysis demonstrated that the pretreatment of gastric epithelial cells with a crosslinking anti-MHC class II IgM blocked the recruitment of FADD to the cell surface. CONCLUSION: The results presented here demonstrate that the ability of MHC class II to modulate gastric epithelial apoptosis is at least partially dependent on its crosslinking. Furthermore, while previous research has demonstrated that MHC class II signaling can be pro-apoptotic during extended ligation, we have shown that the crosslinking of this molecule has anti-apoptotic effects during the earlier time points of H pylori infection. This effect is possibly mediated by the ability of MHC class II to modulate the activation of the pro-apoptotic receptor Fas by blocking the recruitment of the accessory molecule FADD, and this delay in apoptosis induction could allow for prolonged cytokine secretion by H pylori-infected gastric epithelial cells. PMID:16981259

  8. HMGB1 Contributes to the Expression of P-Glycoprotein in Mouse Epileptic Brain through Toll-Like Receptor 4 and Receptor for Advanced Glycation End Products.

    Directory of Open Access Journals (Sweden)

    Yan Chen

    Full Text Available The objective of the present study was to investigate the role of high-mobility group box-1 (HMGB1 in the seizure-induced P-glycoprotein (P-gp overexpression and the underlying mechanism. Kainic acid (KA-induced mouse seizure model was used for in vivo experiments. Male C57BL/6 mice were divided into four groups: normal saline control (NS group, KA-induced epileptic seizure (EP group, and EP group pretreated with HMGB1 (EP+HMGB1 group or BoxA (HMGB1 antagonist, EP+BoxA group. Compared to the NS group, increased levels of HMGB1 and P-gp in the brain were observed in the EP group. Injection of HMGB1 before the induction of KA further increased the expression of P-gp while pre-treatment with BoxA abolished this up-regulation. Next, the regulatory role of HMGB1 and its potential involved signal pathways were investigated in mouse microvascular endothelial bEnd.3 cells in vitro. Cells were treated with HMGB1, HMGB1 plus lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS [toll-like receptor 4 (TLR4 antagonist], HMGB1 plus FPS-ZM1 [receptor for advanced glycation end products (RAGE inhibitor], HMGB1 plus SN50 [nuclear factor-kappa B (NF-κB inhibitor], or vehicle. Treatment with HMGB1 increased the expression levels of P-gp, TLR4, RAGE and the activation of NF-κB in bEnd.3 cells. These effects were inhibited by the pre-treatment with either LPS-RS or FPS-ZM1, and were abolished by the pre-treatment of SN50 or a combination treatment of both LPS-RS and FPS-ZM1. Luciferase reporter assays showed that exogenous expression of NF-κB p65 increased the promoter activity of multidrug resistance 1a (P-gp-encoding gene in endothelial cells. These data indicate that HMGB1 contributes to the overexpression of P-gp in mouse epileptic brain tissues via activation of TLR4/RAGE receptors and the downstream transcription factor NF-κB in brain microvascular endothelial cells.

  9. Nuclear and Membrane Actions of Estrogen Receptor Alpha: Contribution to the Regulation of Energy and Glucose Homeostasis.

    Science.gov (United States)

    Guillaume, Maeva; Montagner, Alexandra; Fontaine, Coralie; Lenfant, Françoise; Arnal, Jean-François; Gourdy, Pierre

    2017-01-01

    Estrogen receptor alpha (ERα) has been demonstrated to play a key role in reproduction but also to exert numerous functions in nonreproductive tissues. Accordingly, ERα is now recognized as a key regulator of energy homeostasis and glucose metabolism and mediates the protective effects of estrogens against obesity and type 2 diabetes. This chapter attempts to summarize our current understanding of the mechanisms of ERα activation and their involvement in the modulation of energy balance and glucose metabolism. We first focus on the experimental studies that constitute the basis of the understanding of ERα as a nuclear receptor and more specifically on the key roles played by its two activation functions (AFs). We depict the consequences of the selective inactivation of these AFs in mouse models, which further underline the prominent role of nuclear ERα in the prevention of obesity and diabetes, as on the reproductive tract and the vascular system. Besides these nuclear actions, a fraction of ERα is associated with the plasma membrane and activates nonnuclear signaling from this site. Such rapid effects, called membrane-initiated steroid signals (MISS), have been characterized in a variety of cell lines and in particular in endothelial cells. The development of selective pharmacological tools that specifically activate MISS as well as the generation of mice expressing an ERα protein impeded for membrane localization has just begun to unravel the physiological role of MISS in vivo and their contribution to ERα-mediated metabolic protection. Finally, we discuss novel perspectives for the design of tissue-selective ER modulators.

  10. Activation of calcitonin gene-related peptide receptor during ozone inhalation contributes to airway epithelial injury and repair.

    Science.gov (United States)

    Oslund, Karen L; Hyde, Dallas M; Putney, Leialoha F; Alfaro, Mario F; Walby, William F; Tyler, Nancy K; Schelegle, Edward S

    2009-10-01

    The authors investigated the importance of the neuropeptide, calcitonin gene-related peptide (CGRP), in epithelial injury, repair, and neutrophil emigration after ozone exposure. Wistar rats were administered either a CGRP-receptor antagonist (CGRP(8-37)) or saline and exposed to 8 hours of 1-ppm ozone or filtered air with an 8-hour postexposure period. Immediately after exposure, ethidium homodimer was instilled into lungs as a marker of necrotic airway epithelial cells. After fixation, airway dissected lung lobes were stained for 5'-bromo-2'-deoxyuridine, a marker of epithelial proliferation. Positive epithelial cells were quantified in specific airway generations. Rats treated with CGRP(8-37) had significantly reduced epithelial injury in terminal bronchioles and reduced epithelial proliferation in proximal airways and terminal bronchioles. Bronchoalveolar lavage and sections of terminal bronchioles showed no significant difference in the number of neutrophils emigrating into airways in CGRP(8-37)-treated rats. The airway epithelial cell line, HBE-1, showed no difference in the number of oxidant stress positive cells during exposure to hydrogen peroxide and a range of CGRP(8-37) doses, demonstrating no antioxidant effect of CGRP(8-37). We conclude that activation of CGRP receptors during ozone inhalation contributes to airway epithelial injury and subsequent epithelial proliferation, a critical component of repair, but does not influence neutrophil emigration into airways.

  11. Contribution of Impulsivity and Serotonin Receptor Neuroadaptations to the Development of an MDMA ('Ecstasy') Substance Use Disorder.

    Science.gov (United States)

    Schenk, Susan; Aronsen, Dane

    As is the case with other drugs of abuse, a proportion of ecstasy users develop symptoms consistent with a substance use disorder (SUD). In this paper, we propose that the pharmacology of MDMA, the primary psychoactive component of ecstasy tablets, changes markedly with repeated exposure and that neuroadaptations in dopamine and serotonin brain systems underlie the shift from MDMA use to MDMA misuse in susceptible subjects. Data from both the human and laboratory animal literature are synthesized to support the idea that (1) MDMA becomes a less efficacious serotonin releaser and a more efficacious dopamine releaser with the development of behaviour consistent with an SUD and (2) that upregulated serotonin receptor mechanisms contribute to the development of the MDMA SUD via dysregulated inhibitory control associated with the trait of impulsivity.

  12. Microglia-Secreted Galectin-3 Acts as a Toll-like Receptor 4 Ligand and Contributes to Microglial Activation

    Directory of Open Access Journals (Sweden)

    Miguel Angel Burguillos

    2015-03-01

    Full Text Available Inflammatory response induced by microglia plays a critical role in the demise of neuronal populations in neuroinflammatory diseases. Although the role of toll-like receptor 4 (TLR4 in microglia’s inflammatory response is fully acknowledged, little is known about endogenous ligands that trigger TLR4 activation. Here, we report that galectin-3 (Gal3 released by microglia acts as an endogenous paracrine TLR4 ligand. Gal3-TLR4 interaction was further confirmed in a murine neuroinflammatory model (intranigral lipopolysaccharide [LPS] injection and in human stroke subjects. Depletion of Gal3 exerted neuroprotective and anti-inflammatory effects following global brain ischemia and in the neuroinflammatory LPS model. These results suggest that Gal3-dependent-TLR4 activation could contribute to sustained microglia activation, prolonging the inflammatory response in the brain.

  13. Protease-activated receptor 1 and 2 contribute to angiotensin II-induced activation of adventitial fibroblasts from rat aorta

    Energy Technology Data Exchange (ETDEWEB)

    He, Rui-Qing; Tang, Xiao-Feng; Zhang, Bao-Li [State Key Laboratory of Medical Genetics, Shanghai Key Laboratory of Hypertension and Department of Hypertension, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (China); Shanghai Institute of Hypertension, Shanghai (China); Li, Xiao-Dong [State Key Laboratory of Medical Genetics, Shanghai Key Laboratory of Hypertension and Department of Hypertension, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (China); Laboratory of Vascular Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai (China); Shanghai Institute of Hypertension, Shanghai (China); Hong, Mo-Na [State Key Laboratory of Medical Genetics, Shanghai Key Laboratory of Hypertension and Department of Hypertension, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (China); Shanghai Institute of Hypertension, Shanghai (China); Chen, Qi-Zhi [Shanghai Institute of Hypertension, Shanghai (China); Han, Wei-Qing, E-mail: whan020@gmail.com [State Key Laboratory of Medical Genetics, Shanghai Key Laboratory of Hypertension and Department of Hypertension, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (China); Laboratory of Vascular Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai (China); Shanghai Institute of Hypertension, Shanghai (China); Gao, Ping-Jin, E-mail: gaopingjin@sibs.ac.cn [State Key Laboratory of Medical Genetics, Shanghai Key Laboratory of Hypertension and Department of Hypertension, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (China); Laboratory of Vascular Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai (China); Shanghai Institute of Hypertension, Shanghai (China)

    2016-04-29

    Adventitial fibroblasts (AFs) can be activated by angiotensin II (Ang II) and exert pro-fibrotic and pro-inflammatory effects in vascular remodeling. Protease-activated receptor (PAR) 1 and 2 play a significant role in fibrogenic and inflammatory diseases. The present study hypothesized that PAR1 and PAR2 are involved in Ang II-induced AF activation and contribute to adventitial remodeling. We found that direct activation of PAR1 and PAR2 with PAR1-AP and PAR2-AP led to AF activation, including proliferation and differentiation of AFs, extracellular matrix synthesis, as well as production of pro-fibrotic cytokine TGF-β and pro-inflammatory cytokines IL-6 and MCP-1. Furthermore, PAR1 and PAR2 mediated Ang II-induced AF activation, since both PAR1 and PAR2 antagonists inhibited Ang II-induced proliferation, migration, differentiation, extracellular matrix synthesis and production of pro-fibrotic and pro-inflammatory cytokines in AFs. Finally, mechanistic study showed that Ang II, via Ang II type I receptor (AT1R), upregulated both PAR1 and PAR2 expression, and transactivated PAR1 and PAR2, as denoted by internalization of both proteins. In conclusion, our results suggest that PAR1 and PAR2 play a critical role in Ang II-induced AF activation, and this may contribute to adventitia-related pathological changes. - Highlights: • Direct activation of PAR1 and PAR2 led to adventitial fibroblast (AF) activation. • PAR1 and PAR2 antagonists attenuated Ang II-induced AF activation. • Ang II induced the upregulation and transactivation of PAR1/PAR2 in AFs.

  14. Protease-activated receptor 1 and 2 contribute to angiotensin II-induced activation of adventitial fibroblasts from rat aorta

    International Nuclear Information System (INIS)

    He, Rui-Qing; Tang, Xiao-Feng; Zhang, Bao-Li; Li, Xiao-Dong; Hong, Mo-Na; Chen, Qi-Zhi; Han, Wei-Qing; Gao, Ping-Jin

    2016-01-01

    Adventitial fibroblasts (AFs) can be activated by angiotensin II (Ang II) and exert pro-fibrotic and pro-inflammatory effects in vascular remodeling. Protease-activated receptor (PAR) 1 and 2 play a significant role in fibrogenic and inflammatory diseases. The present study hypothesized that PAR1 and PAR2 are involved in Ang II-induced AF activation and contribute to adventitial remodeling. We found that direct activation of PAR1 and PAR2 with PAR1-AP and PAR2-AP led to AF activation, including proliferation and differentiation of AFs, extracellular matrix synthesis, as well as production of pro-fibrotic cytokine TGF-β and pro-inflammatory cytokines IL-6 and MCP-1. Furthermore, PAR1 and PAR2 mediated Ang II-induced AF activation, since both PAR1 and PAR2 antagonists inhibited Ang II-induced proliferation, migration, differentiation, extracellular matrix synthesis and production of pro-fibrotic and pro-inflammatory cytokines in AFs. Finally, mechanistic study showed that Ang II, via Ang II type I receptor (AT1R), upregulated both PAR1 and PAR2 expression, and transactivated PAR1 and PAR2, as denoted by internalization of both proteins. In conclusion, our results suggest that PAR1 and PAR2 play a critical role in Ang II-induced AF activation, and this may contribute to adventitia-related pathological changes. - Highlights: • Direct activation of PAR1 and PAR2 led to adventitial fibroblast (AF) activation. • PAR1 and PAR2 antagonists attenuated Ang II-induced AF activation. • Ang II induced the upregulation and transactivation of PAR1/PAR2 in AFs.

  15. Cannabinoid CB2 Receptors Contribute to Upregulation of β-endorphin in Inflamed Skin Tissues by Electroacupuncture

    Directory of Open Access Journals (Sweden)

    Su Tang-feng

    2011-12-01

    Full Text Available Abstract Background Electroacupuncture (EA can produce analgesia by increasing the β-endorphin level and activation of peripheral μ-opioid receptors in inflamed tissues. Endogenous cannabinoids and peripheral cannabinoid CB2 receptors (CB2Rs are also involved in the antinociceptive effect of EA on inflammatory pain. However, little is known about how peripheral CB2Rs interact with the endogenous opioid system at the inflammatory site and how this interaction contributes to the antinociceptive effect of EA on inflammatory pain. In this study, we determined the role of peripheral CB2Rs in the effects of EA on the expression of β-endorphin in inflamed skin tissues and inflammatory pain. Results Inflammatory pain was induced by injection of complete Freund's adjuvant into the left hindpaw of rats. Thermal hyperalgesia was tested with a radiant heat stimulus, and mechanical allodynia was quantified using von Frey filaments. The mRNA level of POMC and protein level of β-endorphin were quantified by real-time PCR and Western blotting, respectively. The β-endorphin-containing keratinocytes and immune cells in the inflamed skin tissues were detected by double-immunofluorescence labeling. The CB2R agonist AM1241 or EA significantly reduced thermal hyperalgesia and mechanical allodynia, whereas the selective μ-opioid receptor antagonist β-funaltrexamine significantly attenuated the antinociceptive effect produced by them. AM1241 or EA significantly increased the mRNA level of POMC and the protein level of β-endorphin in inflamed skin tissues, and these effects were significantly attenuated by pretreatment with the CB2R antagonist AM630. AM1241 or EA also significantly increased the percentage of β-endorphin-immunoreactive keratinocytes, macrophages, and T-lymphocytes in inflamed skin tissues, and these effects were blocked by AM630. Conclusions EA and CB2R stimulation reduce inflammatory pain through activation of μ-opioid receptors. EA increases

  16. Fc receptorsubunits with both polar or non-polar amino acids at position of T22 are capable of restoring surface expression of the high-affinity IgE receptor and degranulation in γ subunit-deficient rat basophilic leukemia cells

    Czech Academy of Sciences Publication Activity Database

    Rashid, A.; Housden, J.E.; Helm, B.A.; Dráber, Petr

    2013-01-01

    Roč. 53, č. 3 (2013), s. 270-273 ISSN 0161-5890 R&D Projects: GA MŠk LD12073; GA ČR GA301/09/1826; GA ČR GAP302/10/1759; GA ČR(CZ) GBP302/12/G101; GA MŠk 1M0506 Grant - others:European Cooperation in Science and Technology(XE) BM1007; AV ČR(CZ) M200520901 Institutional research plan: CEZ:AV0Z50520514 Keywords : allergy * high-affinity IgE receptor * plasma membrane * transmembrane signaling * 3-helix assembly model Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.003, year: 2013

  17. Contribution of Hippocampal 5-HT3 Receptors in Hippocampal Autophagy and Extinction of Conditioned Fear Responses after a Single Prolonged Stress Exposure in Rats.

    Science.gov (United States)

    Wu, Zhong-Min; Yang, Li-Hua; Cui, Rong; Ni, Gui-Lian; Wu, Feng-Tian; Liang, Yong

    2017-05-01

    One of the hypotheses about the pathogenesis of posttraumatic stress disorder (PTSD) is the dysfunction of serotonin (5-HT) neurotransmission. While certain 5-HT receptor subtypes are likely critical for the symptoms of PTSD, few studies have examined the role of 5-HT 3 receptor in the development of PTSD, even though 5-HT 3 receptor is critical for contextual fear extinction and anxiety-like behavior. Therefore, we hypothesized that stimulation of 5-HT 3 receptor in the dorsal hippocampus (DH) could prevent hippocampal autophagy and the development of PTSD-like behavior in animals. To this end, we infused SR57227, selective 5-HT 3 agonist, into the DH after a single prolonged stress (SPS) treatment in rats. Three weeks later, we evaluated the effects of this pharmacological treatment on anxiety-related behaviors and extinction of contextual fear memory. We also accessed hippocampal autophagy and the expression of 5-HT 3A subunit, Beclin-1, LC3-I, and LC3-II in the DH. We found that SPS treatment did not alter anxiety-related behaviors but prolonged the extinction of contextual fear memory, and such a behavioral phenomenon was correlated with increased hippocampal autophagy, decreased 5-HT 3A expression, and increased expression of Beclin-1 and LC3-II/LC3-I ratio in the DH. Furthermore, intraDH infusions of SR57227 dose-dependently promoted the extinction of contextual fear memory, prevented hippocampal autophagy, and decreased expression of Beclin-1 and LC3-II/LC3-I ratio in the DH. These results indicated that 5-HT 3 receptor in the hippocampus may play a critical role in the pathogenesis of hippocampal autophagy, and is likely involved in the pathophysiology of PTSD.

  18. Family environment and adult resilience: contributions of positive parenting and the oxytocin receptor gene

    Directory of Open Access Journals (Sweden)

    Bekh Bradley

    2013-09-01

    Full Text Available Background: Abundant research shows that childhood adversity increases the risk for adult psychopathology while research on influences of positive family environment on risk for psychopathology is limited. Similarly, a growing body of research examines genetic and gene by environment predictors of psychopathology, yet such research on predictors of resilience is sparse. Objectives: We examined the role of positive factors in childhood family environment (CFE and the OXTR rs53576 genotype in predicting levels of adult resilient coping and positive affect. We also examined whether the relationship between positive factors in the CFEs and adult resilient coping and positive affect varied across OXTR rs53576 genotype. Methods: We gathered self-report data on childhood environment, trauma history, and adult resilience and positive affect in a sample of 971 African American adults. Results: We found that positive CFE was positively associated with higher levels of resilient coping and positive affect in adulthood after controlling for childhood maltreatment, other trauma, and symptoms of posttraumatic stress disorder. We did not find a direct effect of OXTR 53576 on a combined resilient coping/positive-affect-dependent variable, but we did find an interaction of OXTR rs53576 with family environment. Conclusions: Our data suggest that even in the face of adversity, positive aspects of the family environment may contribute to resilience. These results highlight the importance of considering protective developmental experiences and the interaction of such experiences with genetic variants in risk and resilience research.For the abstract or full text in other languages, please see Supplementary files under Article Tools online

  19. The cannabinoid receptor CB1 contributes to the development of ectopic lesions in a mouse model of endometriosis.

    Science.gov (United States)

    Sanchez, Ana-Maria; Quattrone, Federica; Pannese, Maria; Ulisse, Adele; Candiani, Massimo; Diaz-Alonso, Javier; Velasco, Guillermo; Panina-Bordignon, Paola

    2017-01-01

    Does signaling via the cannabinoid (CB 1 ) receptor play a role in the pathogenesis of endometriosis in a mouse model? Mice treated with a CB 1 agonist developed larger ectopic lesions, while less severe lesions developed in the absence of functional CB 1 expression. The expression of components of the endocannabinoid system has been demonstrated in both mouse and human uteri. CB 1 receptors are expressed in human epithelial and stromal cell lines derived from eutopic endometrium and deep infiltrating endometriosis nodules. This was a randomized study in a mouse model of endometriosis. In a first set of experiments, mice with endometriosis were treated with the CB 1 receptor agonist methanandamide (MET) (5 mg/kg, n = 20) on Days 1-5 and 8-12. In a second set of experiments, endometriosis development was evaluated in CB 1 -/- mice and in their wild-type (WT) littermates. Endometriosis-like lesions were induced in Balb/c and C57/Bl6 mice. Two weeks after disease induction, the lesions were counted, measured and either included for immunohistochemistry analysis or frozen for gene expression profiling by semi-quantitative real-time PCR. To limit the role of chance, the experiments were conducted under standardized laboratory conditions with appropriate controls. The lesion total volume was significantly higher in MET-treated compared with vehicle-treated mice (P endometriosis in a mouse model. However, the relative contribution of the CB 1 -mediated signaling pathways active in inflammatory, uterine and peritoneal cells remains to be ascertained. Since the study was performed in a mouse model, the significance of the findings in the human system warrants further investigation. Clarifying the function and regulation of CB 1 and its molecular interactions with endogenous ligands, and how endocannabinoids levels are regulated in women with endometriosis, represent critical areas of research for the potential development of a novel medical treatment of the disease. A

  20. The Contribution of Peroxisome Proliferator-Activated Receptor Alpha to the Relationship Between Toxicokinetics and Toxicodynamics of Trichloroethylene.

    Science.gov (United States)

    Yoo, Hong Sik; Cichocki, Joseph A; Kim, Sungkyoon; Venkatratnam, Abhishek; Iwata, Yasuhiro; Kosyk, Oksana; Bodnar, Wanda; Sweet, Stephen; Knap, Anthony; Wade, Terry; Campbell, Jerry; Clewell, Harvey J; Melnyk, Stepan B; Chiu, Weihsueh A; Rusyn, Ivan

    2015-10-01

    Exposure to the ubiquitous environmental contaminant trichloroethylene (TCE) is associated with cancer and non-cancer toxicity in both humans and rodents. Peroxisome proliferator-activated receptor-alpha (PPARα) is thought to be playing a role in liver toxicity in rodents through activation of the receptor by the TCE metabolite trichloroacetic acid (TCA). However, most studies using genetically altered mice have not assessed the potential for PPARα to alter TCE toxicokinetics, which may lead to differences in TCA internal doses and hence confound inferences as to the role of PPARα in TCE toxicity. To address this gap, male and female wild type (129S1/SvImJ), Pparα-null, and humanized PPARα (hPPARα) mice were exposed intragastrically to 400 mg/kg TCE in single-dose (2, 5 and 12 h) and repeat-dose (5 days/week, 4 weeks) studies. Interestingly, following either a single- or repeat-dose exposure to TCE, levels of TCA in liver and kidney were lower in Pparα-null and hPPARα mice as compared with those in wild type mice. Levels of trichloroethanol (TCOH) were similar in all strains. TCE-exposed male mice consistently had higher levels of TCA and TCOH in all tissues compared with females. Additionally, in both single- and repeat-dose studies, a similar degree of induction of PPARα-responsive genes was observed in liver and kidney of hPPARα and wild type mice, despite the difference in hepatic and renal TCA levels. Additional sex- and strain-dependent effects were observed in the liver, including hepatocyte proliferation and oxidative stress, which were not dependent on TCA or TCOH levels. These data demonstrate that PPARα status affects the levels of the putative PPARα agonist TCA following TCE exposure. Therefore, interpretations of studies using Pparα-null and hPPARα mice need to consider the potential contribution of genotype-dependent toxicokinetics to observed differences in toxicity, rather than attributing such differences only to receptor

  1. Type-I Insulin-Like Growth Factor Receptor (IGF1R)-Estrogen Receptor (ER) Crosstalk Contributes to Antiestrogen Therapy Resistance in Breast Cancer Cells

    Science.gov (United States)

    2013-02-01

    vitro have downregulated J GF1R making antibodies directed agai nst th is receptor ineffective. Inhlbition of IH may be necessary to manage ...monoclonal antibody to insulin-like growth factor receptor 1. J Clin Oncol 2009;27:580Q-7. 31. Drury s. Detre s. Leary A, Salter J, Reis-Filho J

  2. Arabidopsis TNL-WRKY domain receptor RRS1 contributes to temperature-conditioned RPS4 auto-immunity

    Directory of Open Access Journals (Sweden)

    Katharina eHeidrich

    2013-10-01

    Full Text Available In plant effector-triggered immunity (ETI, intracellular nucleotide binding-leucine rich repeat (NLR receptors are activated by specific pathogen effectors. The Arabidopsis TIR (Toll Interleukin1 receptor domain-NLR (denoted TNL gene pair, RPS4 and RRS1, confers resistance to Pseudomonas syringae pv tomato (Pst strain DC3000 expressing the Type III-secreted effector, AvrRps4. Nuclear accumulation of AvrRps4, RPS4 and the TNL resistance regulator EDS1 is necessary for ETI. RRS1 possesses a C-terminal ‘WRKY’ transcription factor DNA binding domain suggesting that important RPS4/RRS1 recognition and/or resistance signaling events occur at the nuclear chromatin. In Arabidopsis accession Ws-0, the RPS4Ws/RRS1Ws allelic pair governs resistance to Pst/AvrRps4 accompanied by host programmed cell death (pcd. In accession Col-0, RPS4Col/RRS1Col effectively limits Pst/AvrRps4 growth without pcd. Constitutive expression of HA-StrepII tagged RPS4Col (in a 35S:RPS4-HS line confers temperature conditioned EDS1-dependent auto-immunity. Here we show that a high (28oC, non-permissive to moderate (19oC, permissive temperature shift of 35S:RPS4-HS plants can be used to follow defense-related transcriptional dynamics without a pathogen effector trigger. By comparing responses of 35S:RPS4-HS with 35S:RPS4-HS rrs1-11 and 35S:RPS4-HS eds1-2 mutants, we establish that RPS4Col auto-immunity depends entirely on EDS1 and partially on RRS1Col. Examination of gene expression microarray data over 24h after temperature shift reveals a mainly quantitative RRS1Col contribution to up- or down-regulation of a small subset of RPS4Col-reprogrammed, EDS1-dependent genes. We find significant over-representation of WRKY transcription factor binding W-box cis-elements within the promoters of these genes. Our data show that RRS1Col contributes to temperature-conditioned RPS4Col auto-immunity and are consistent with activated RPS4Col engaging RRS1Col for resistance signaling.

  3. DHEAS increases levels of GluR2/3 and GluR2, AMPA receptor subunits, in C57BL/6 mice hippocampus El DHEAS incrementa la expresión de GluR2/3 y GLUR2 del receptor AMPA en el hipocampo de ratones C57/BL6

    Directory of Open Access Journals (Sweden)

    Diego Sepúlveda Falla

    2010-05-01

    Full Text Available

    Dehydroepiandrosterone sulfate (DHEA-S is a neurosteroid that has effects such as neuromodulator of synaptic transmission and neuroprotection. The specific signaling pathways for these effects are not elucidated yet. Given that, some neurosteroids act through the activation of ionotropic glutamate receptors, therefore the effect of DHEA-S on the subunits GluR2  and GluR3 of the AMPA receptor was evaluated.  Either DHEA-S or a control substance was administered to C57/BL6 mice. Subunit expression of the AMPA receptor was analyzed by Western blotting.

     

     

    Results show that long-term DHEA-S administration to C57/BL6 mice, increases the protein levels of the subunits GluR2 and GluR2/3 of the AMPA receptors located in the hippocampus.

  4. Identification and elucidation of anthropogenic source contribution in PM10 pollutant: Insight gain from dispersion and receptor models.

    Science.gov (United States)

    Roy, Debananda; Singh, Gurdeep; Yadav, Pankaj

    2016-10-01

    Source apportionment study of PM 10 (Particulate Matter) in a critically polluted area of Jharia coalfield, India has been carried out using Dispersion model, Principle Component Analysis (PCA) and Chemical Mass Balance (CMB) techniques. Dispersion model Atmospheric Dispersion Model (AERMOD) was introduced to simplify the complexity of sources in Jharia coalfield. PCA and CMB analysis indicates that monitoring stations near the mining area were mainly affected by the emission from open coal mining and its associated activities such as coal transportation, loading and unloading of coal. Mine fire emission also contributed a considerable amount of particulate matters in monitoring stations. Locations in the city area were mostly affected by vehicular, Liquid Petroleum Gas (LPG) & Diesel Generator (DG) set emissions, residential, and commercial activities. The experimental data sampling and their analysis could aid understanding how dispersion based model technique along with receptor model based concept can be strategically used for quantitative analysis of Natural and Anthropogenic sources of PM 10 . Copyright © 2016. Published by Elsevier B.V.

  5. Toll-Like Receptor 4 in Bone Marrow-Derived Cells Contributes to the Progression of Diabetic Retinopathy

    Directory of Open Access Journals (Sweden)

    Hui Wang

    2014-01-01

    Full Text Available Diabetic retinopathy (DR is a major microvascular complication in diabetics, and its mechanism is not fully understood. Toll-like receptor 4 (TLR4 plays a pivotal role in the maintenance of the inflammatory state during DR, and the deletion of TLR4 eventually alleviates the diabetic inflammatory state. To further elucidate the mechanism of DR, we used bone marrow transplantation to establish reciprocal chimeric animals of TLR4 mutant mice and TLR4 WT mice combined with diabetes mellitus (DM induction by streptozotocin (STZ treatment to identify the role of TLR4 in different cell types in the development of the proinflammatory state during DR. TLR4 mutation did not block the occurrence of high blood glucose after STZ injection compared with WT mice but did alleviate the progression of DR and alter the expression of the small vessel proliferation-related genes, vascular endothelial growth factor (VEGF, and hypoxia inducible factor-1α (HIF-1α. Grafting bone marrow-derived cells from TLR4 WT mice into TLR4 mutant mice increased the levels of TNF-α, IL-1β, and MIP-2 and increased the damage to the retina. Similarly, VEGF and HIF-1α expression were restored by the bone marrow transplantation. These findings identify an essential role for TLR4 in bone marrow-derived cells contributing to the progression of DR.

  6. INTRINSIC REGULATION OF HEMOGLOBIN EXPRESSION BY VARIABLE SUBUNIT INTERFACE STRENGTHS

    Science.gov (United States)

    Manning, James M.; Popowicz, Anthony M.; Padovan, Julio C.; Chait, Brian T.; Manning, Lois R.

    2012-01-01

    SUMMARY The expression of the six types of human hemoglobin subunits over time is currently considered to be regulated mainly by transcription factors that bind to upstream control regions of the gene (the “extrinsic” component of regulation). Here we describe how subunit pairing and further assembly to tetramers in the liganded state is influenced by the affinity of subunits for one another (the “intrinsic” component of regulation). The adult hemoglobin dimers have the strongest subunit interfaces and the embryonic hemoglobins are the weakest with fetal hemoglobins of intermediate strength, corresponding to the temporal order of their expression. These variable subunit binding strengths and the attenuating effects of acetylation contribute to the differences with which these hemoglobin types form functional O2-binding tetramers consistent with gene switching. PMID:22129306

  7. Typical and Atypical Antipsychotic Drugs Increase Extracellular Histamine Levels in the Rat Medial Prefrontal Cortex: Contribution of Histamine H1 Receptor Blockade

    Directory of Open Access Journals (Sweden)

    Kjell A Svensson

    2012-05-01

    Full Text Available Atypical antipsychotics such as clozapine and olanzapine have been shown to enhance histamine turnover and this effect has been hypothesized to contribute to their improved therapeutic profile compared to typical antipsychotics. In the present study, we examined the effects of antipsychotic drugs on histamine (HA efflux in the mPFC of the rat by means of in vivo microdialysis and sought to differentiate the receptor mechanisms which underlie such effects. Olanzapine and clozapine increased mPFC HA efflux in a dose related manner. Increased HA efflux was also observed after quetiapine, chlorpromazine and perphenazine treatment. We found no effect of the selective 5-HT2A antagonist MDL100907, 5-HT2c antagonist SB242084 or the 5-HT6 antagonist Ro 04-6790 on mPFC HA efflux. HA efflux was increased following treatment with selective H1 receptor antagonists pyrilamine, diphenhydramine and triprolidine, the H3 receptor antagonist ciproxifan and the mixed 5HT2A/H1 receptor antagonist ketanserin. The potential novel antipsychotic drug FMPD, which has a lower affinity at H1 receptors than olanzapine, did not affect HA efflux. Similarly, other antipsychotics with lower H1 receptor affinity (risperidone, aripiprazole and haloperidol were also without effect on HA efflux. Perfusion of clozapine and pyrilamine into the TMN, but not the mPFC, increased local HA efflux. Finally, HA efflux after antipsychotic treatment was significantly correlated with affinity at H1 receptors whereas 9 other receptors, including 5-HT2A, were not. These results demonstrate that both typical and atypical antipsychotics increase mPFC histamine efflux and this effect may be mediated via antagonism of histamine H1 receptors.

  8. The disintegrin and metalloproteinase ADAM12 contributes to TGF-beta signaling through interaction with the type II receptor

    DEFF Research Database (Denmark)

    Atfi, Azeddine; Dumont, Emmanuelle; Colland, Frédéric

    2007-01-01

    Transforming growth factor-beta (TGF-beta) regulates a wide variety of biological processes through two types of Ser/Thr transmembrane receptors: the TGF-beta type I receptor and the TGF-beta type II receptor (TbetaRII). Upon ligand binding, TGF-beta type I receptor activated by TbetaRII propagat......RII protein presumably by suppressing the association of TbetaRII with Smad7. These results define ADAM12 as a new partner of TbetaRII that facilitates its trafficking to early endosomes in which activation of the Smad pathway is initiated....

  9. receptores

    Directory of Open Access Journals (Sweden)

    Salete Regina Daronco Benetti

    2006-01-01

    Full Text Available Se trata de un estudio etnográfico, que tuvo lo objetivo de interpretar el sistema de conocimiento y del significado atribuidos a la sangre referente a la transfusión sanguínea por los donadores y receptores de un banco de sangre. Para la colecta de las informaciones se observaron los participantes y la entrevista etnográfica se realizó el análisis de dominio, taxonómicos y temáticos. Los dominios culturales fueron: la sangre es vida: fuente de vida y alimento valioso; creencias religiosas: fuentes simbólicas de apoyos; donación sanguínea: un gesto colaborador que exige cuidarse, gratifica y trae felicidad; donación sanguínea: fuente simbólica de inseguridad; estar enfermo es una condición para realizar transfusión sanguínea; transfusión sanguínea: esperanza de vida; Creencias populares: transfusión sanguínea como riesgo para la salud; donadores de sangre: personas benditas; donar y recibir sangre: como significado de felicidad. Temática: “líquido precioso que origina, sostiene, modifica la vida, provoca miedo e inseguridad”.

  10. Pharmacological evidence that a failure to recruit NMDA receptors contributes to impaired fear extinction retention in adolescent rats.

    Science.gov (United States)

    Baker, Kathryn D; Richardson, Rick

    2017-09-01

    Adolescents, both humans and rodents, exhibit a marked impairment in extinction of fear relative to younger and older groups which could be caused by a failure to efficiently recruit NMDA receptors (NMDARs) in adolescence. It is well-established that systemic administration of NMDAR antagonists (e.g., MK801) before extinction training impairs the retention of extinction in adult and juvenile rodents, but it is unknown whether this is also the case for adolescents. Therefore, in the present study we investigated the effect of pharmacologically manipulating the NMDAR on extinction retention in adolescent rats. When extinction retention is typically impaired (i.e., after one session of extinction training) adolescent male rats given d-cycloserine (a partial NMDAR agonist) showed enhanced extinction retention relative to saline-treated animals while animals given MK801 (a non-competitive antagonist) did not exhibit any further impairment of extinction retention relative to the controls. In a further two experiments we demonstrated that when two sessions of extinction training separated by either 4 or 24h intervals were given to adolescent rats, saline-treated animals exhibited good extinction retention and the animals given MK801 before the second session exhibited impaired extinction retention. These findings suggest that extinction in adolescence does not initially involve NMDARs and this is a likely mechanism that contributes to the impaired fear inhibition observed at this age. However, NMDARs appear to be recruited with extended extinction training or after administration of a partial agonist, both of which lead to effective extinction retention. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Adult forebrain NMDA receptors gate social motivation and social memory.

    Science.gov (United States)

    Jacobs, Stephanie; Tsien, Joe Z

    2017-02-01

    Motivation to engage in social interaction is critical to ensure normal social behaviors, whereas dysregulation in social motivation can contribute to psychiatric diseases such as schizophrenia, autism, social anxiety disorders and post-traumatic stress disorder (PTSD). While dopamine is well known to regulate motivation, its downstream targets are poorly understood. Given the fact that the dopamine 1 (D1) receptors are often physically coupled with the NMDA receptors, we hypothesize that the NMDA receptor activity in the adult forebrain principal neurons are crucial not only for learning and memory, but also for the proper gating of social motivation. Here, we tested this hypothesis by examining sociability and social memory in inducible forebrain-specific NR1 knockout mice. These mice are ideal for exploring the role of the NR1 subunit in social behavior because the NR1 subunit can be selectively knocked out after the critical developmental period, in which NR1 is required for normal development. We found that the inducible deletion of the NMDA receptors prior to behavioral assays impaired, not only object and social recognition memory tests, but also resulted in profound deficits in social motivation. Mice with ablated NR1 subunits in the forebrain demonstrated significant decreases in sociability compared to their wild type counterparts. These results suggest that in addition to its crucial role in learning and memory, the NMDA receptors in the adult forebrain principal neurons gate social motivation, independent of neuronal development. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. A Special Extract of Bacopa monnieri (CDRI-08 Restores Learning and Memory by Upregulating Expression of the NMDA Receptor Subunit GluN2B in the Brain of Scopolamine-Induced Amnesic Mice

    Directory of Open Access Journals (Sweden)

    Rakesh Rai

    2015-01-01

    Full Text Available In the present communication, we have investigated effects of the CDRI-08, a well characterized extract of Bacopa monnieri, on expression of the GluN2B subunit of NMDAR in various brain regions of the scopolamine-induced amnesic mice. Our behavioral data reveal that scopolamine-treated amnesic mice exhibit significant decline in the spatial memory compared to the normal control mice. Our RT-PCR and immunoblotting data revealed that the scopolamine treatment resulted in a significant downregulation of the NMDAR GluN2B subunit expression in prefrontal cortex and hippocampus. Our enzyme assay data revealed that scopolamine caused a significant increase in the acetylcholinesterase activity in both the brain regions. Further, oral administration of the CDRI-08 to scopolamine-treated amnesic mice restored the spatial memory which was found to be associated with significant upregulation of the GluN2B subunit expression and decline in the acetylcholinesterase activity in prefrontal cortex as well as hippocampus towards their levels in the normal control mice. Our study provides the evidence for the mechanism underlying role of the Bacopa monnieri extract (CDRI-08 in restoring spatial memory in amnesic mice, which may have therapeutic implications.

  13. Highly conserved small subunit residues influence rubisco large subunit catalysis.

    Science.gov (United States)

    Genkov, Todor; Spreitzer, Robert J

    2009-10-30

    The chloroplast enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthetic CO(2) fixation. With a deeper understanding of its structure-function relationships and competitive inhibition by O(2), it may be possible to engineer an increase in agricultural productivity and renewable energy. The chloroplast-encoded large subunits form the active site, but the nuclear-encoded small subunits can also influence catalytic efficiency and CO(2)/O(2) specificity. To further define the role of the small subunit in Rubisco function, the 10 most conserved residues in all small subunits were substituted with alanine by transformation of a Chlamydomonas reinhardtii mutant that lacks the small subunit gene family. All the mutant strains were able to grow photosynthetically, indicating that none of the residues is essential for function. Three of the substitutions have little or no effect (S16A, P19A, and E92A), one primarily affects holoenzyme stability (L18A), and the remainder affect catalysis with or without some level of associated structural instability (Y32A, E43A, W73A, L78A, P79A, and F81A). Y32A and E43A cause decreases in CO(2)/O(2) specificity. Based on the x-ray crystal structure of Chlamydomonas Rubisco, all but one (Glu-92) of the conserved residues are in contact with large subunits and cluster near the amino- or carboxyl-terminal ends of large subunit alpha-helix 8, which is a structural element of the alpha/beta-barrel active site. Small subunit residues Glu-43 and Trp-73 identify a possible structural connection between active site alpha-helix 8 and the highly variable small subunit loop between beta-strands A and B, which can also influence Rubisco CO(2)/O(2) specificity.

  14. AGE and their receptor RAGE in systemic autoimmune diseases : An inflammation propagating factor contributing to accelerated atherosclerosis

    NARCIS (Netherlands)

    Nienhuis, Hans L. A.; Westra, Johanna; Smit, Andries J.; Limburg, Pieter C.; Kallenberg, Cees G. M.; Bijl, Marc

    2009-01-01

    Systemic autoimmune diseases are associated with inflammation, and oxidative stress favouring the formation of advanced glycation endproducts (AGE), able to modulate cellular functions by activation of receptor for advanced glycation endproducts (RAGE). As RAGE expression is increased in an

  15. Three alpha-subunits of heterotrimeric G proteins and an adenylyl cyclase have distinct roles in fruiting body development in the homothallic fungus Sordaria macrospora.

    Science.gov (United States)

    Kamerewerd, Jens; Jansson, Malin; Nowrousian, Minou; Pöggeler, Stefanie; Kück, Ulrich

    2008-09-01

    Sordaria macrospora, a self-fertile filamentous ascomycete, carries genes encoding three different alpha-subunits of heterotrimeric G proteins (gsa, G protein Sordaria alpha subunit). We generated knockout strains for all three gsa genes (Deltagsa1, Deltagsa2, and Deltagsa3) as well as all combinations of double mutants. Phenotypic analysis of single and double mutants showed that the genes for Galpha-subunits have distinct roles in the sexual life cycle. While single mutants show some reduction of fertility, double mutants Deltagsa1Deltagsa2 and Deltagsa1Deltagsa3 are completely sterile. To test whether the pheromone receptors PRE1 and PRE2 mediate signaling via distinct Galpha-subunits, two recently generated Deltapre strains were crossed with all Deltagsa strains. Analyses of the corresponding double mutants revealed that compared to GSA2, GSA1 is a more predominant regulator of a signal transduction cascade downstream of the pheromone receptors and that GSA3 is involved in another signaling pathway that also contributes to fruiting body development and fertility. We further isolated the gene encoding adenylyl cyclase (AC) (sac1) for construction of a knockout strain. Analyses of the three DeltagsaDeltasac1 double mutants and one Deltagsa2Deltagsa3Deltasac1 triple mutant indicate that SAC1 acts downstream of GSA3, parallel to a GSA1-GSA2-mediated signaling pathway. In addition, the function of STE12 and PRO41, two presumptive signaling components, was investigated in diverse double mutants lacking those developmental genes in combination with the gsa genes. This analysis was further completed by expression studies of the ste12 and pro41 transcripts in wild-type and mutant strains. From the sum of all our data, we propose a model for how different Galpha-subunits interact with pheromone receptors, adenylyl cyclase, and STE12 and thus cooperatively regulate sexual development in S. macrospora.

  16. Protein-tyrosine Phosphatase SHP2 Contributes to GDNF Neurotrophic Activity through Direct Binding to Phospho-Tyr687 in the RET Receptor Tyrosine Kinase*

    Science.gov (United States)

    Perrinjaquet, Maurice; Vilar, Marçal; Ibáñez, Carlos F.

    2010-01-01

    The signaling mechanisms by which neurotrophic receptors regulate neuronal survival and axonal growth are still incompletely understood. In the receptor tyrosine kinase RET, a receptor for GDNF (glial cell line-derived neurotrophic factor), the functions of the majority of tyrosine residues that become phosphorylated are still unknown. Here we have identified the protein-tyrosine phosphatase SHP2 as a novel direct interactor of RET and the first effector known to bind to phosphorylated Tyr687 in the juxtamembrane region of the receptor. We show that SHP2 is recruited to RET upon ligand binding in a cooperative fashion, such that both interaction with Tyr687 and association with components of the Tyr1062 signaling complex are required for stable recruitment of SHP2 to the receptor. SHP2 recruitment contributes to the ability of RET to activate the PI3K/AKT pathway and promote survival and neurite outgrowth in primary neurons. Furthermore, we find that activation of protein kinase A (PKA) by forskolin reduces the recruitment of SHP2 to RET and negatively affects ligand-mediated neurite outgrowth. In agreement with this, mutation of Ser696, a known PKA phosphorylation site in RET, enhances SHP2 binding to the receptor and eliminates the effect of forskolin on ligand-induced outgrowth. Together, these findings establish SHP2 as a novel positive regulator of the neurotrophic activities of RET and reveal Tyr687 as a critical platform for integration of RET and PKA signals. We anticipate that several other phosphotyrosines of unknown function in neuronal receptor tyrosine kinases will also support similar regulatory functions. PMID:20682772

  17. Protein-tyrosine phosphatase SHP2 contributes to GDNF neurotrophic activity through direct binding to phospho-Tyr687 in the RET receptor tyrosine kinase.

    Science.gov (United States)

    Perrinjaquet, Maurice; Vilar, Marçal; Ibáñez, Carlos F

    2010-10-08

    The signaling mechanisms by which neurotrophic receptors regulate neuronal survival and axonal growth are still incompletely understood. In the receptor tyrosine kinase RET, a receptor for GDNF (glial cell line-derived neurotrophic factor), the functions of the majority of tyrosine residues that become phosphorylated are still unknown. Here we have identified the protein-tyrosine phosphatase SHP2 as a novel direct interactor of RET and the first effector known to bind to phosphorylated Tyr(687) in the juxtamembrane region of the receptor. We show that SHP2 is recruited to RET upon ligand binding in a cooperative fashion, such that both interaction with Tyr(687) and association with components of the Tyr(1062) signaling complex are required for stable recruitment of SHP2 to the receptor. SHP2 recruitment contributes to the ability of RET to activate the PI3K/AKT pathway and promote survival and neurite outgrowth in primary neurons. Furthermore, we find that activation of protein kinase A (PKA) by forskolin reduces the recruitment of SHP2 to RET and negatively affects ligand-mediated neurite outgrowth. In agreement with this, mutation of Ser(696), a known PKA phosphorylation site in RET, enhances SHP2 binding to the receptor and eliminates the effect of forskolin on ligand-induced outgrowth. Together, these findings establish SHP2 as a novel positive regulator of the neurotrophic activities of RET and reveal Tyr(687) as a critical platform for integration of RET and PKA signals. We anticipate that several other phosphotyrosines of unknown function in neuronal receptor tyrosine kinases will also support similar regulatory functions.

  18. Toll-like receptor 4 (TLR4) impairs nitric oxide contributing to Angiotensin II-induced cavernosal dysfunction.

    Science.gov (United States)

    Nunes, Kenia P; Bomfim, Gisele F; Toque, Haroldo A; Szasz, Theodora; Clinton Webb, R

    2017-12-15

    Angiotensin II (AngII), a corpus cavernosum (CC) constrictor peptide, modulates Toll like receptor (TLR) expression, a key element of the innate immune system, contributing to impaired vascular function in pathological conditions. However, it is unknown whether TLR4 is involved in AngII-induced erectile dysfunction. In this study, we investigated whether TLR4 plays a role in cavernosal dysfunction caused by AngII upregulation. Cavernosal smooth muscle cells (CSMC) from C57/BL6 mice were treated with AngII (0.1μM) or bacterial LPS (50ng/ml) for 12-24h and TLR4 expression was assessed. Mice were infused with AngII (90ng/min, 28days) and treated with anti-TLR4 antibody (0.1mg/daily, i.p.) for the last 14days of the treatment. CC tissue was used for functional studies and for Western blotting. Nitric Oxide Synthase (NOS) activity was measured by conversion of [ 3 H]-l-arginine to [ 3 H]-l-citrulline, systemic TNF-α levels by ELISA, and reactive oxygen species (ROS) by immunofluorescence. We report upregulation of TLR4 in CSMC following AngII or LPS stimulation. In AngII-infused mice, chronic treatment with anti-TLR4 antibody (28±2.1%) attenuates adrenergic CC contraction, which also ameliorates nitrergic (68.90±0.21 vs. 51.07±0.63, 8Hz, AngII-infused mice treated vs. non-treated). Decreased endothelial NOS expression, reduced NOS activity, and augmented levels of TNF-α, and ROS were found following AngII-infusion. These alterations were prevented, or at least decreased by anti-TLR4 antibody treatment. Inhibition of TLR4 ameliorates AngII-impaired cavernosal relaxation, decreases TNF-α levels, and restores NO bioavailability, demonstrating that TLR4 partly mediates AngII-induced cavernosal dysfunction. Copyright © 2017. Published by Elsevier Inc.

  19. Clinical utility of circulating anti-N-methyl-d-aspartate receptor subunits NR2A/B antibody for the diagnosis of neuropsychiatric syndromes in systemic lupus erythematosus and Sjögren's syndrome: An updated meta-analysis.

    Science.gov (United States)

    Tay, Sen Hee; Fairhurst, Anna-Marie; Mak, Anselm

    2017-02-01

    Neuropsychiatric (NP) events are found in patients with rheumatic diseases, commonly in systemic lupus erythematosus (SLE) and Sjögren's syndrome (SS). The standard nomenclature and case definitions for 19 NPSLE syndromes by the American College of Rheumatology (ACR) Committee on Research cover a wide range of NP events seen in both SLE and SS. Despite advances in the understanding of SLE and SS, NP syndromes continue to pose diagnostic challenges. Correct attribution of NP events is critical in determining the correct treatment and prognosis. Anti-N-methyl- d -aspartate receptor subunits NR2A/B (anti-NR2A/B) antibodies have been demonstrated in the sera of SLE and SS patients and have been associated with collective or specific NP syndromes, though not consistently. Interpretation of anti-NR2A/B antibody data in the medical literature is rendered difficult by small sample size of patient groups. By combining different studies to generate a pooled effect size, a meta-analysis can increase the power to detect differences in the presence or absence of NP syndromes. Hence, we set out to perform a meta-analysis to assess the association between anti-NR2A/B antibodies and NP syndromes in SLE and SS. A literature search was conducted using PubMed and other databases from inception to June 2016. We abstracted data relating to anti-NR2A/B antibodies from the identified studies. The random effects model was used to calculate overall combined odds ratio (OD) with its corresponding 95% confidence interval (CI) to evaluate the relationship between anti-NR2A/B antibodies and NP syndromes in SLE and SS patients with and without NP events. We also included our own cohort of 57 SLE patients fulfilling the ACR 1997 revised classification criteria and 58 healthy controls (HCs). In total, 17 studies with data on anti-NR2A/B antibodies in 2212 SLE patients, 66 SS patients, 99 disease controls (DCs) (e.g. antiphospholipid syndrome, myasthenia gravis and autoimmune polyendocrine

  20. Aberrant Receptor Internalization and Enhanced FRS2-dependent Signaling Contribute to the Transforming Activity of the Fibroblast Growth Factor Receptor 2 IIIb C3 Isoform*

    OpenAIRE

    Cha, Jiyoung Y.; Maddileti, Savitri; Mitin, Natalia; Harden, T. Kendall; Der, Channing J.

    2009-01-01

    Alternative splice variants of fibroblast growth factor receptor 2 (FGFR2) IIIb, designated C1, C2, and C3, possess progressive reduction in their cytoplasmic carboxyl termini (822, 788, and 769 residues, respectively), with preferential expression of the C2 and C3 isoforms in human cancers. We determined that the progressive deletion of carboxyl-terminal sequences correlated with increasing transforming potency. The highly transforming C3 variant lacks five tyrosine r...

  1. Aberrant Receptor Internalization and Enhanced FRS2-dependent Signaling Contribute to the Transforming Activity of the Fibroblast Growth Factor Receptor 2 IIIb C3 Isoform*

    Science.gov (United States)

    Cha, Jiyoung Y.; Maddileti, Savitri; Mitin, Natalia; Harden, T. Kendall; Der, Channing J.

    2009-01-01

    Alternative splice variants of fibroblast growth factor receptor 2 (FGFR2) IIIb, designated C1, C2, and C3, possess progressive reduction in their cytoplasmic carboxyl termini (822, 788, and 769 residues, respectively), with preferential expression of the C2 and C3 isoforms in human cancers. We determined that the progressive deletion of carboxyl-terminal sequences correlated with increasing transforming potency. The highly transforming C3 variant lacks five tyrosine residues present in C1, and we determined that the loss of Tyr-770 alone enhanced FGFR2 IIIb C1 transforming activity. Because Tyr-770 may compose a putative YXXL sorting motif, we hypothesized that loss of Tyr-770 in the 770YXXL motif may cause disruption of FGFR2 IIIb C1 internalization and enhance transforming activity. Surprisingly, we found that mutation of Leu-773 but not Tyr-770 impaired receptor internalization and increased receptor stability and activation. Interestingly, concurrent mutations of Tyr-770 and Leu-773 caused 2-fold higher transforming activity than caused by the Y770F or L773A single mutations, suggesting loss of Tyr and Leu residues of the 770YXXL773 motif enhances FGFR2 IIIb transforming activity by distinct mechanisms. We also determined that loss of Tyr-770 caused persistent activation of FRS2 by enhancing FRS2 binding to FGFR2 IIIb. Furthermore, we found that FRS2 binding to FGFR2 IIIb is required for increased FRS2 tyrosine phosphorylation and enhanced transforming activity by Y770F mutation. Our data support a dual mechanism where deletion of the 770YXXL773 motif promotes FGFR2 IIIb C3 transforming activity by causing aberrant receptor recycling and stability and persistent FRS2-dependent signaling. PMID:19103595

  2. The role of oestrogen receptor {alpha} in human thyroid cancer: contributions from coregulatory proteins and the tyrosine kinase receptor HER2.

    LENUS (Irish Health Repository)

    Kavanagh, Dara O

    2012-02-01

    Epidemiological, clinical, and molecular studies suggest a role for oestrogen in thyroid cancer. How oestrogen mediates its effects and the consequence of it on clinical outcome has not been fully elucidated. The participation of coregulatory proteins in modulating oestrogen receptor (ER) function and input of crosstalk with the tyrosine kinase receptor HER2 was investigated. Oestrogen induced cell proliferation in the follicular thyroid cancer (FTC)-133 cells, but not in the anaplastic 8305C cell line. Knockdown of the coactivator steroid receptor coactivator (SRC)-1 inhibited FTC-133 basal, but not oestrogen induced, cell proliferation. Oestrogen also increased protein expression of SRC-1 and the ER target gene cyclin D1 in the FTC-133 cell line. ERalpha, ERbeta, the coregulatory proteins SRC-1 and nuclear corepressor (NCoR), and the tyrosine kinase receptor HER2 were localised by immunohistochemistry and immnofluorescence in paraffin-embedded tissue from thyroid tumour patients (n=111). ERalpha was colocalised with both SRC-1 and NCoR to the nuclei of the tumour epithelial cells. Expression of ERalpha and NCoR was found predominantly in non-anaplastic tumours and was significantly associated with well-differentiated tumours and reduced incidence of disease recurrence. In non-anaplastic tumours, HER2 was significantly associated with SRC-1, and these proteins were associated with poorly differentiated tumours, capsular invasion and disease recurrence. Totally, 87% of anaplastic tumours were positive for SRC-1. Kaplan-Meier estimates of disease-free survival indicated that in thyroid cancer, SRC-1 strongly correlates with reduced disease-free survival (P<0.001), whereas NCoR predicted increased survival (P<0.001). These data suggest opposing roles for the coregulators SRC-1 and NCoR in thyroid tumour progression.

  3. The extracellular loop 2 (ECL2 of the human histamine H4 receptor substantially contributes to ligand binding and constitutive activity.

    Directory of Open Access Journals (Sweden)

    David Wifling

    Full Text Available In contrast to the corresponding mouse and rat orthologs, the human histamine H4 receptor (hH4R shows extraordinarily high constitutive activity. In the extracellular loop (ECL, replacement of F169 by V as in the mouse H4R significantly reduced constitutive activity. Stabilization of the inactive state was even more pronounced for a double mutant, in which, in addition to F169V, S179 in the ligand binding site was replaced by M. To study the role of the FF motif in ECL2, we generated the hH4R-F168A mutant. The receptor was co-expressed in Sf9 insect cells with the G-protein subunits Gαi2 and Gβ1γ2, and the membranes were studied in [3H]histamine binding and functional [35S]GTPγS assays. The potency of various ligands at the hH4R-F168A mutant decreased compared to the wild-type hH4R, for example by 30- and more than 100-fold in case of the H4R agonist UR-PI376 and histamine, respectively. The high constitutive activity of the hH4R was completely lost in the hH4R-F168A mutant, as reflected by neutral antagonism of thioperamide, a full inverse agonist at the wild-type hH4R. By analogy, JNJ7777120 was a partial inverse agonist at the hH4R, but a partial agonist at the hH4R-F168A mutant, again demonstrating the decrease in constitutive activity due to F168A mutation. Thus, F168 was proven to play a key role not only in ligand binding and potency, but also in the high constitutive activity of the hH4R.

  4. The Contribution of Proteinase-Activated Receptors to Intracellular Signaling, Transcellular Transport and Autophagy in Alzheimer´s Disease

    Czech Academy of Sciences Publication Activity Database

    Matěj, R.; Rohan, Z.; Holada, K.; Olejár, Tomáš

    2015-01-01

    Roč. 12, č. 1 (2015), s. 2-12 ISSN 1567-2050 Institutional support: RVO:67985823 Keywords : Alzheimer ´s Disease * autophagy * proteinase-activated receptors Subject RIV: EA - Cell Biology Impact factor: 3.145, year: 2015

  5. Down-regulation of NR2B receptors partially contributes to analgesic effects of Gentiopicroside in persistent inflammatory pain.

    Science.gov (United States)

    Chen, Lei; Liu, Jin-cheng; Zhang, Xiao-nan; Guo, Yan-yan; Xu, Zhao-hui; Cao, Wei; Sun, Xiao-li; Sun, Wen-ji; Zhao, Ming-Gao

    2008-06-01

    Gentiopicroside is one of the secoiridoid compound isolated from Gentiana lutea. It exhibits analgesic activities in the mice. The anterior cingulate cortex (ACC) is a forebrain structure known for its roles in pain transmission and modulation. Painful stimuli potentiate the prefrontal synaptic transmission and induce glutamate NMDA NR2B receptor expression in the ACC. But little is known about Gentiopicroside on the persistent inflammatory pain and chronic pain-induced synaptic transmission changes in the ACC. The present study was undertaken to investigate its analgesic activities and central synaptic modulation to the peripheral painful inflammation. Gentiopicroside produced significant analgesic effects against persistent inflammatory pain stimuli in mice. Systemic administration of Gentiopicroside significantly reversed NR2B over-expression during the chronic phases of persistent inflammation caused by hind-paw administration of complete Freunds adjuvant (CFA) in mice. Whole-cell patch clamp recordings revealed that Gentiopicroside significantly reduced NR2B receptors mediated postsynaptic currents in the ACC. Our findings provide strong evidence that analgesic effects of Gentiopicroside involve down-regulation of NR2B receptors in the ACC to persistent inflammatory pain.

  6. The Oxytocin Receptor (OXTR) Contributes to Prosocial Fund Allocations in the Dictator Game and the Social Value Orientations Task

    Science.gov (United States)

    Israel, Salomon; Lerer, Elad; Shalev, Idan; Uzefovsky, Florina; Riebold, Mathias; Laiba, Efrat; Bachner-Melman, Rachel; Maril, Anat; Bornstein, Gary; Knafo, Ariel; Ebstein, Richard P.

    2009-01-01

    Background Economic games observe social decision making in the laboratory that involves real money payoffs. Previously we have shown that allocation of funds in the Dictator Game (DG), a paradigm that illustrates costly altruistic behavior, is partially determined by promoter-region repeat region variants in the arginine vasopressin 1a receptor gene (AVPR1a). In the current investigation, the gene encoding the related oxytocin receptor (OXTR) was tested for association with the DG and a related paradigm, the Social Values Orientation (SVO) task. Methodology/Principal Findings Association (101 male and 102 female students) using a robust-family based test between 15 single tagging SNPs (htSNPs) across the OXTR was demonstrated with both the DG and SVO. Three htSNPs across the gene region showed significant association with both of the two games. The most significant association was observed with rs1042778 (p = 0.001). Haplotype analysis also showed significant associations for both DG and SVO. Following permutation test adjustment, significance was observed for 2–5 locus haplotypes (pprosocial decision making converges with a large body of animal research showing that oxytocin is an important social hormone across vertebrates including Homo sapiens. Individual differences in prosocial behavior have been shown by twin studies to have a substantial genetic basis and the current investigation demonstrates that common variants in the oxytocin receptor gene, an important element of mammalian social circuitry, underlie such individual differences. PMID:19461999

  7. Liposome-Based Adjuvants for Subunit Vaccines: Formulation Strategies for Subunit Antigens and Immunostimulators

    Directory of Open Access Journals (Sweden)

    Signe Tandrup Schmidt

    2016-03-01

    Full Text Available The development of subunit vaccines has become very attractive in recent years due to their superior safety profiles as compared to traditional vaccines based on live attenuated or whole inactivated pathogens, and there is an unmet medical need for improved vaccines and vaccines against pathogens for which no effective vaccines exist. The subunit vaccine technology exploits pathogen subunits as antigens, e.g., recombinant proteins or synthetic peptides, allowing for highly specific immune responses against the pathogens. However, such antigens are usually not sufficiently immunogenic to induce protective immunity, and they are often combined with adjuvants to ensure robust immune responses. Adjuvants are capable of enhancing and/or modulating immune responses by exposing antigens to antigen-presenting cells (APCs concomitantly with conferring immune activation signals. Few adjuvant systems have been licensed for use in human vaccines, and they mainly stimulate humoral immunity. Thus, there is an unmet demand for the development of safe and efficient adjuvant systems that can also stimulate cell-mediated immunity (CMI. Adjuvants constitute a heterogeneous group of compounds, which can broadly be classified into delivery systems or immunostimulators. Liposomes are versatile delivery systems for antigens, and they can carefully be customized towards desired immune profiles by combining them with immunostimulators and optimizing their composition, physicochemical properties and antigen-loading mode. Immunostimulators represent highly diverse classes of molecules, e.g., lipids, nucleic acids, proteins and peptides, and they are ligands for pattern-recognition receptors (PRRs, which are differentially expressed on APC subsets. Different formulation strategies might thus be required for incorporation of immunostimulators and antigens, respectively, into liposomes, and the choice of immunostimulator should ideally be based on knowledge regarding the

  8. Normalized Synergy Predicts That CD8 Co-Receptor Contribution to T Cell Receptor (TCR and pMHC Binding Decreases As TCR Affinity Increases in Human Viral-Specific T Cells

    Directory of Open Access Journals (Sweden)

    Chad M. Williams

    2017-07-01

    Full Text Available The discovery of naturally occurring T cell receptors (TCRs that confer specific, high-affinity recognition of pathogen and cancer-associated antigens remains a major goal in cellular immunotherapies. The contribution of the CD8 co-receptor to the interaction between the TCR and peptide-bound major histocompatibility complex (pMHC has previously been correlated with the activation and responsiveness of CD8+ T cells. However, these studies have been limited to model systems of genetically engineered hybridoma TCRs or transgenic mouse TCRs against either a single epitope or an array of altered peptide ligands. CD8 contribution in a native human antigen-specific T cell response remains elusive. Here, using Hepatitis C Virus-specific precursor CTLs spanning a large range of TCR affinities, we discovered that the functional responsiveness of any given TCR correlated with the contribution of CD8 to TCR/pMHC binding. Furthermore, we found that CD8 contribution to TCR/pMHC binding in the two-dimensional (2D system was more accurately reflected by normalized synergy (CD8 cooperation normalized by total TCR/pMHC bonds rather than synergy (total CD8 cooperation alone. While synergy showed an increasing trend with TCR affinity, normalized synergy was demonstrated to decrease with the increase of TCR affinity. Critically, normalized synergy was shown to correlate with CTL functionality and peptide sensitivity, corroborating three-dimensional (3D analysis of CD8 contribution with respect to TCR affinity. In addition, we identified TCRs that were independent of CD8 for TCR/pMHC binding. Our results resolve the current discrepancy between 2D and 3D analysis on CD8 contribution to TCR/pMHC binding, and demonstrate that naturally occurring high-affinity TCRs are more capable of CD8-independent interactions that yield greater functional responsiveness even with CD8 blocking. Taken together, our data suggest that addition of the normalized synergy parameter to our

  9. Contribution of altered signal transduction associated to glutamate receptors in brain to the neurological alterations of hepatic encephalopathy

    Institute of Scientific and Technical Information of China (English)

    Vicente Felipo

    2006-01-01

    Patients with liver disease may present hepatic encephalopathy (HE), a complex neuropsychiatric syndrome covering a wide range of neurological alterations,including cognitive and motor disturbances. HE reduces the quality of life of the patients and is associated with poor prognosis. In the worse cases HE may lead to coma or death.The mechanisms leading to HE which are not well known are being studied using animal models. The neurological alterations in HE are a consequence of impaired cerebral function mainly due to alterations in neurotransmission. We review here some studies indicating that alterations in neurotransmission associated to different types of glutamate receptors are responsible for some of the cognitive and motor alterations present in HE.These studies show that the function of the signal transduction pathway glutamate-nitric oxide-cGMP associated to the NMDA type of glutamate receptors is impaired in brain in vivo in HE animal models as well as in brain of patients died of HE. Activation of NMDA receptors in brain activates this pathway and increases cGMP. In animal models of HE this increase in cGMP induced by activation of NMDA receptors is reduced,which is responsible for the impairment in learning ability in these animal models. Increasing cGMP by pharmacological means restores learning ability in rats with HE and may be a new therapeutic approach to improve cognitive function in patients with HE.However, it is necessary to previously assess the possible secondary effects.Patients with HE may present psychomotor slowing,hypokinesia and bradykinesia. Animal models of HE also show hypolocomotion. It has been shown in rats with HE that hypolocomotion is due to excessive activation of metabotropic glutamate receptors (mGluRs) in substantia nigra pars reticulata. Blocking mGluR1 in this brain area normalizes motor activity in the rats, suggesting that a similar treatment for patients with HE could be useful to treat psychomotor slowing and

  10. Do motor control genes contribute to interindividual variability in decreased movement in patients with pain?

    Directory of Open Access Journals (Sweden)

    Mishra Bikash K

    2007-07-01

    Full Text Available Abstract Background Because excessive reduction in activities after back injury may impair recovery, it is important to understand and address the factors contributing to the variability in motor responses to pain. The current dominant theory is the "fear-avoidance model", in which the some patients' heightened fears of further injury cause them to avoid movement. We propose that in addition to psychological factors, neurochemical variants in the circuits controlling movement and their modification by pain may contribute to this variability. A systematic search of the motor research literature and genetic databases yielded a prioritized list of polymorphic motor control candidate genes. We demonstrate an analytic method that we applied to 14 of these genes in 290 patients with acute sciatica, whose reduction in movement was estimated by items from the Roland-Morris Disability Questionnaire. Results We genotyped a total of 121 single nucleotide polymorphisms (SNPs in 14 of these genes, which code for the dopamine D2 receptor, GTP cyclohydrolase I, glycine receptor α1 subunit, GABA-A receptor α2 subunit, GABA-A receptor β1 subunit, α-adrenergic 1C, 2A, and 2C receptors, serotonin 1A and 2A receptors, cannabinoid CB-1 receptor, M1 muscarinic receptor, and the tyrosine hydroxylase, and tachykinin precursor-1 molecules. No SNP showed a significant association with the movement score after a Bonferroni correction for the 14 genes tested. Haplotype analysis of one of the blocks in the GABA-A receptor β1 subunit showed that a haplotype of 11% frequency was associated with less limitation of movement at a nominal significance level value (p = 0.0025 almost strong enough to correct for testing 22 haplotype blocks. Conclusion If confirmed, the current results may suggest that a common haplotype in the GABA-A β1 subunit acts like an "endogenous muscle relaxant" in an individual with subacute sciatica. Similar methods might be applied a larger set of

  11. The role of GluN2A and GluN2B NMDA receptor subunits in AgRP and POMC neurons on body weight and glucose homeostasis

    Directory of Open Access Journals (Sweden)

    Aykut Üner

    2015-10-01

    Conclusions: GluN2B-containing NMDA receptors in AgRP neurons play a critical role in central control of body weight homeostasis and blood glucose balance via mechanisms that likely involve regulation of AgRP neuronal survival and structure, and modulation of hypothalamic leptin action.

  12. Structural characteristics of anabolic androgenic steroids contributing to binding to the androgen receptor and to their anabolic and androgenic activities. Applied modifications in the steroidal structure.

    Science.gov (United States)

    Fragkaki, A G; Angelis, Y S; Koupparis, M; Tsantili-Kakoulidou, A; Kokotos, G; Georgakopoulos, C

    2009-02-01

    Anabolic androgenic steroids (AAS) are synthetic derivatives of testosterone introduced for therapeutic purposes providing enhanced anabolic potency with reduced androgenic effects. Androgens mediate their action through their binding to the androgen receptor (AR) which is mainly expressed in androgen target tissues, such as the prostate, skeletal muscle, liver and central nervous system. This paper reviews some of the wide spectrum of testosterone and synthetic AAS structure modifications related to the intended enhancement in anabolic activity. The structural features of steroids necessary for effective binding to the AR and those which contribute to the stipulation of the androgenic and anabolic activities are also presented.

  13. Contribution of G protein-coupled estrogen receptor 1 (GPER) to 17β-estradiol-induced developmental toxicity in zebrafish.

    Science.gov (United States)

    Diamante, Graciel; Menjivar-Cervantes, Norma; Leung, Man Sin; Volz, David C; Schlenk, Daniel

    2017-05-01

    Exposure to 17β-estradiol (E2) influences the regulation of multiple signaling pathways, and E2-mediated disruption of signaling events during early development can lead to malformations such as cardiac defects. In this study, we investigated the potential role of the G-protein estrogen receptor 1 (GPER) in E2-induced developmental toxicity. Zebrafish embryos were exposed to E2 from 2h post fertilization (hpf) to 76 hpf with subsequent transcriptional measurements of heart and neural crest derivatives expressed 2 (hand2), leucine rich repeat containing 10 (lrrc10), and gper at 12, 28 and 76 hpf. Alteration in the expression of lrrc10, hand2 and gper was observed at 12 hpf and 76 hpf, but not at 28 hpf. Expression of these genes was also altered after exposure to G1 (a GPER agonist) at 76 hpf. Expression of lrrc10, hand2 and gper all coincided with the formation of cardiac edema at 76 hpf as well as other developmental abnormalities. While co-exposure of G1 with G36 (a GPER antagonist) rescued G1-induced abnormalities and altered gene expression, co-exposure of E2 with G36, or ICI 182,780 (an estrogen receptor antagonist) did not rescue E2-induced cardiac deformities or gene expression. In addition, no effects on the concentrations of downstream ER and GPER signaling molecules (cAMP or calcium) were observed in embryo homogenates after E2 treatment. These data suggest that the impacts of E2 on embryonic development at this stage are complex and may involve multiple receptor and/or signaling pathways. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. The oxytocin receptor (OXTR contributes to prosocial fund allocations in the dictator game and the social value orientations task.

    Directory of Open Access Journals (Sweden)

    Salomon Israel

    Full Text Available BACKGROUND: Economic games observe social decision making in the laboratory that involves real money payoffs. Previously we have shown that allocation of funds in the Dictator Game (DG, a paradigm that illustrates costly altruistic behavior, is partially determined by promoter-region repeat region variants in the arginine vasopressin 1a receptor gene (AVPR1a. In the current investigation, the gene encoding the related oxytocin receptor (OXTR was tested for association with the DG and a related paradigm, the Social Values Orientation (SVO task. METHODOLOGY/PRINCIPAL FINDINGS: Association (101 male and 102 female students using a robust-family based test between 15 single tagging SNPs (htSNPs across the OXTR was demonstrated with both the DG and SVO. Three htSNPs across the gene region showed significant association with both of the two games. The most significant association was observed with rs1042778 (p = 0.001. Haplotype analysis also showed significant associations for both DG and SVO. Following permutation test adjustment, significance was observed for 2-5 locus haplotypes (p<0.05. A second sample of 98 female subjects was subsequently and independently recruited to play the dictator game and was genotyped for the three significant SNPs found in the first sample. The rs1042778 SNP was shown to be significant for the second sample as well (p = 0.004, Fisher's exact test. CONCLUSIONS: The demonstration that genetic polymorphisms for the OXTR are associated with human prosocial decision making converges with a large body of animal research showing that oxytocin is an important social hormone across vertebrates including Homo sapiens. Individual differences in prosocial behavior have been shown by twin studies to have a substantial genetic basis and the current investigation demonstrates that common variants in the oxytocin receptor gene, an important element of mammalian social circuitry, underlie such individual differences.

  15. The cysteinyl leukotriene 2 receptor contributes to all-trans retinoic acid-induced differentiation of colon cancer cells

    International Nuclear Information System (INIS)

    Bengtsson, Astrid M; Jönsson, Gunilla; Magnusson, Cecilia; Salim, Tavga; Axelsson, Cecilia; Sjölander, Anita

    2013-01-01

    Cysteinyl leukotrienes (CysLTs) are potent pro-inflammatory mediators that are increased in samples from patients with inflammatory bowel diseases (IBDs). Individuals with IBDs have enhanced susceptibility to colon carcinogenesis. In colorectal cancer, the balance between the pro-mitogenic cysteinyl leukotriene 1 receptor (CysLT 1 R) and the differentiation-promoting cysteinyl leukotriene 2 receptor (CysLT 2 R) is lost. Further, our previous data indicate that patients with high CysLT 1 R and low CysLT 2 R expression have a poor prognosis. In this study, we examined whether the balance between CysLT 1 R and CysLT 2 R could be restored by treatment with the cancer chemopreventive agent all-trans retinoic acid (ATRA). To determine the effect of ATRA on CysLT 2 R promoter activation, mRNA level, and protein level, we performed luciferase gene reporter assays, real-time polymerase chain reactions, and Western blots in colon cancer cell lines under various conditions. ATRA treatment induces CysLT 2 R mRNA and protein expression without affecting CysLT 1 R levels. Experiments using siRNA and mutant cell lines indicate that the up-regulation is retinoic acid receptor (RAR) dependent. Interestingly, ATRA also up-regulates mRNA expression of leukotriene C 4 synthase, the enzyme responsible for the production of the ligand for CysLT 2 R. Importantly, ATRA-induced differentiation of colorectal cancer cells as shown by increased expression of MUC-2 and production of alkaline phosphatase, both of which could be reduced by a CysLT 2 R-specific inhibitor. This study identifies a novel mechanism of action for ATRA in colorectal cancer cell differentiation and demonstrates that retinoids can have anti-tumorigenic effects through their action on the cysteinyl leukotriene pathway

  16. Contribution of non-genetic factors to dopamine and serotonin receptor availability in the adult human brain

    DEFF Research Database (Denmark)

    Borg, J; Cervenka, S; Kuja-Halkola, R

    2016-01-01

    The dopamine (DA) and serotonin (5-HT) neurotransmission systems are of fundamental importance for normal brain function and serve as targets for treatment of major neuropsychiatric disorders. Despite central interest for these neurotransmission systems in psychiatry research, little is known about...... and environmental factors, respectively, on dopaminergic and serotonergic markers in the living human brain. Eleven monozygotic and 10 dizygotic healthy male twin pairs were examined with PET and [(11)C]raclopride binding to the D2- and D3-dopamine receptor and [(11)C]WAY100635 binding to the serotonin 5-HT1A...

  17. Activity-dependent shedding of the NMDA receptor glycine binding site by matrix metalloproteinase 3: a PUTATIVE mechanism of postsynaptic plasticity.

    Directory of Open Access Journals (Sweden)

    Thorsten Pauly

    Full Text Available Functional and structural alterations of clustered postsynaptic ligand gated ion channels in neuronal cells are thought to contribute to synaptic plasticity and memory formation in the human brain. Here, we describe a novel molecular mechanism for structural alterations of NR1 subunits of the NMDA receptor. In cultured rat spinal cord neurons, chronic NMDA receptor stimulation induces disappearance of extracellular epitopes of NMDA receptor NR1 subunits, which was prevented by inhibiting matrix metalloproteinases (MMPs. Immunoblotting revealed the digestion of solubilized NR1 subunits by MMP-3 and identified a fragment of about 60 kDa as MMPs-activity-dependent cleavage product of the NR1 subunit in cultured neurons. The expression of MMP-3 in the spinal cord culture was shown by immunoblotting and immunofluorescence microscopy. Recombinant NR1 glycine binding protein was used to identify MMP-3 cleavage sites within the extracellular S1 and S2-domains. N-terminal sequencing and site-directed mutagenesis revealed S542 and L790 as two putative major MMP-3 cleavage sites of the NR1 subunit. In conclusion, our data indicate that MMPs, and in particular MMP-3, are involved in the activity dependent alteration of NMDA receptor structure at postsynaptic membrane specializations in the CNS.

  18. Activity-dependent shedding of the NMDA receptor glycine binding site by matrix metalloproteinase 3: a PUTATIVE mechanism of postsynaptic plasticity.

    Science.gov (United States)

    Pauly, Thorsten; Ratliff, Miriam; Pietrowski, Eweline; Neugebauer, Rainer; Schlicksupp, Andrea; Kirsch, Joachim; Kuhse, Jochen

    2008-07-16

    Functional and structural alterations of clustered postsynaptic ligand gated ion channels in neuronal cells are thought to contribute to synaptic plasticity and memory formation in the human brain. Here, we describe a novel molecular mechanism for structural alterations of NR1 subunits of the NMDA receptor. In cultured rat spinal cord neurons, chronic NMDA receptor stimulation induces disappearance of extracellular epitopes of NMDA receptor NR1 subunits, which was prevented by inhibiting matrix metalloproteinases (MMPs). Immunoblotting revealed the digestion of solubilized NR1 subunits by MMP-3 and identified a fragment of about 60 kDa as MMPs-activity-dependent cleavage product of the NR1 subunit in cultured neurons. The expression of MMP-3 in the spinal cord culture was shown by immunoblotting and immunofluorescence microscopy. Recombinant NR1 glycine binding protein was used to identify MMP-3 cleavage sites within the extracellular S1 and S2-domains. N-terminal sequencing and site-directed mutagenesis revealed S542 and L790 as two putative major MMP-3 cleavage sites of the NR1 subunit. In conclusion, our data indicate that MMPs, and in particular MMP-3, are involved in the activity dependent alteration of NMDA receptor structure at postsynaptic membrane specializations in the CNS.

  19. The Neurokinin-1 Receptor Contributes to the Early Phase of Lipopolysaccharide-Induced Fever via Stimulation of Peripheral Cyclooxygenase-2 Protein Expression in Mice

    Directory of Open Access Journals (Sweden)

    Eszter Pakai

    2018-02-01

    Full Text Available Neurokinin (NK signaling is involved in various inflammatory processes. A common manifestation of systemic inflammation is fever, which is usually induced in animal models with the administration of bacterial lipopolysaccharide (LPS. A role for the NK1 receptor was shown in LPS-induced fever, but the underlying mechanisms of how the NK1 receptor contributes to febrile response, especially in the early phase, have remained unknown. We administered LPS (120 µg/kg, intraperitoneally to mice with the Tacr1 gene, i.e., the gene encoding the NK1 receptor, either present (Tacr1+/+ or absent (Tacr1−/− and measured their thermoregulatory responses, serum cytokine levels, tissue cyclooxygenase-2 (COX-2 expression, and prostaglandin (PG E2 concentration. We found that the LPS-induced febrile response was attenuated in Tacr1−/− compared to their Tacr1+/+ littermates starting from 40 min postinfusion. The febrigenic effect of intracerebroventricularly administered PGE2 was not suppressed in the Tacr1−/− mice. Serum concentration of pyrogenic cytokines did not differ between Tacr1−/− and Tacr1+/+ at 40 min post-LPS infusion. Administration of LPS resulted in amplification of COX-2 mRNA expression in the lungs, liver, and brain of the mice, which was statistically indistinguishable between the genotypes. In contrast, the LPS-induced augmentation of COX-2 protein expression was attenuated in the lungs and tended to be suppressed in the liver of Tacr1−/− mice compared with Tacr1+/+ mice. The Tacr1+/+ mice responded to LPS with a significant surge of PGE2 production in the lungs, whereas Tacr1−/− mice did not. In conclusion, the NK1 receptor is necessary for normal fever genesis. Our results suggest that the NK1 receptor contributes to the early phase of LPS-induced fever by enhancing COX-2 protein expression in the periphery. These findings advance the understanding of the crosstalk between NK signaling and the “cytokine-COX-2

  20. Heterotrimeric G protein subunits are located on rat liver endosomes

    Directory of Open Access Journals (Sweden)

    Van Dyke Rebecca W

    2004-01-01

    Full Text Available Abstract Background Rat liver endosomes contain activated insulin receptors and downstream signal transduction molecules. We undertook these studies to determine whether endosomes also contain heterotrimeric G proteins that may be involved in signal transduction from G protein-coupled receptors. Results By Western blotting Gsα, Giα1,2, Giα3 and Gβ were enriched in both canalicular (CM and basolateral (BLM membranes but also readily detectable on three types of purified rat liver endosomes in the order recycling receptor compartment (RRC > compartment for uncoupling of receptor and ligand (CURL > multivesicular bodies (MVB >> purified secondary lysosomes. Western blotting with antibodies to Na, K-ATPase and to other proteins associated with plasma membranes and intracellular organelles indicated this was not due to contamination of endosome preparations by CM or BLM. Adenylate cyclase (AC was also identified on purified CM, BLM, RRC, CURL and MVB. Percoll gradient fractionation of liver postnuclear supernatants demonstrated co-occurrence of endosomes and heterotrimeric G protein subunits in fractions with little plasma membrane markers. By confocal microscopy, punctate staining for Gsα, Giα3 and Gβ corresponded to punctate areas of endocytosed Texas red-dextran in hepatocytes from control and cholera toxin-treated livers. Conclusion We conclude that heterotrimeric G protein subunits as well as AC likely traffic into hepatocytes on endosome membranes, possibly generating downstream signals spatially separate from signalling generated at the plasma membrane, analogous to the role(s of internalized insulin receptors.

  1. Evaluation of peptide designing strategy against subunit reassociation in mucin 1: A steered molecular dynamics approach.

    Directory of Open Access Journals (Sweden)

    J Lesitha Jeeva Kumari

    Full Text Available Subunit reassociation in mucin 1, a breast cancer tumor marker, is reported as one of the critical factors for its cytoplasmic activation. Inhibition of its heterodimeric association would therefore result in loss of its function and alter disease progression. The present study aimed at evaluating peptide inhibitor designing strategies that may serve as antagonist against this receptor-ligand alliance. Several peptides and their derivatives were designed based on native residues, subunit interface, hydrogen bonding and secondary structure. Docking studies with the peptides were carried on the receptor subunit and their binding affinities were evaluated using steered molecular dynamics simulation and umbrella sampling. Our results showed that among all the different classes of peptides evaluated, the receptor based peptide showed the highest binding affinity. This result was concurrent with the experimental observation that the receptor-ligand alliance in mucin 1 is highly specific. Our results also show that peptide ligand against this subunit association is only stabilized through native residue inter-protein interaction irrespective of the peptide structure, peptide length and number of hydrogen bonds. Consistency in binding affinity, pull force and free energy barrier was observed with only the receptor derived peptides which resulted in favorable interprotein interactions at the interface. Several observations were made and discussed which will eventually lead to designing efficient peptide inhibitors against mucin 1 heterodimeric subunit reassociation.

  2. Beta-adrenergic receptors in the lateral nucleus of the amygdala contribute to the acquisition but not the consolidation of auditory fear conditioning.

    Science.gov (United States)

    Bush, David E A; Caparosa, Ellen M; Gekker, Anna; Ledoux, Joseph

    2010-01-01

    Beta-adrenergic receptors (βARs) have long been associated with fear disorders and with learning and memory. However, the contribution of these receptors to Pavlovian fear conditioning, a leading behavioral model for studying fear learning and memory, is still poorly understood. The aim of this study was to investigate the involvement of βAR activation in the acquisition, consolidation and expression of fear conditioning. We focused on manipulations of βARs in the lateral nucleus of the amygdala (LA) because of the well-established contribution of this area to fear conditioning. Specifically, we tested the effects of intra-LA microinfusions of the βAR antagonist, propranolol, on learning and memory for auditory Pavlovian fear conditioning in rats. Pre-training propranolol infusions disrupted the initial acquisition, short-term memory (STM), and long-term memory (LTM) for fear conditioning, but infusions immediately after training had no effect. Further, infusion of propranolol prior to testing fear responses did not affect fear memory expression. These findings indicate that amygdala βARs are important for the acquisition but not the consolidation of fear conditioning.

  3. Brain-derived neurotrophic factor (BDNF) in the rostral anterior cingulate cortex (rACC) contributes to neuropathic spontaneous pain-related aversion via NR2B receptors.

    Science.gov (United States)

    Zhang, Le; Wang, Gongming; Ma, Jinben; Liu, Chengxiao; Liu, Xijiang; Zhan, Yufeng; Zhang, Mengyuan

    2016-10-01

    The rostral anterior cingulate cortex (rACC) plays an important role in pain affect. Previous investigations have reported that the rACC mediates the negative affective component of inflammatory pain and contributed to the aversive state of nerve injury-induced neuropathic pain. Brain-derived neurotrophic factor (BDNF), an activity-dependent neuromodulator in the adult brain, is believed to play a role in the development and maintenance of inflammatory and neuropathic pain in the spinal cord. However, whether and how BDNF in the rACC regulates pain-related aversion due to peripheral nerve injury is largely unknown. Behaviorally, using conditioned place preference (CPP) training in rats, which is thought to reveal spontaneous pain-related aversion, we found that CPP was acquired following spinal clonidine in rats with partial sciatic nerve transection. Importantly, BDNF was upregulated within the rACC in of rats with nerve injury and enhanced the CPP acquisition, while a local injection of a BDNF-tropomyosin receptor kinase B (TrkB) antagonist into the rACC completely blocked this process. Finally, we demonstrated that the BDNF/TrkB pathway exerted its function by activating the NR2B receptor, which is widely accepted to be a crucial factor contributing to pain affect. In conclusion, our results demonstrate that the BDNF/TrkB-mediated signaling pathway in the rACC is involved in the development of neuropathic spontaneous pain-related aversion and that this process is dependent upon activation of NR2B receptors. These findings suggest that suppression of the BDNF-related signaling pathway in the rACC may provide a novel strategy to overcome pain-related aversion. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. TFIIH subunit alterations causing xeroderma pigmentosum and trichothiodystrophy specifically disturb several steps during transcription.

    Science.gov (United States)

    Singh, Amita; Compe, Emanuel; Le May, Nicolas; Egly, Jean-Marc

    2015-02-05

    Mutations in genes encoding the ERCC3 (XPB), ERCC2 (XPD), and GTF2H5 (p8 or TTD-A) subunits of the transcription and DNA-repair factor TFIIH lead to three autosomal-recessive disorders: xeroderma pigmentosum (XP), XP associated with Cockayne syndrome (XP/CS), and trichothiodystrophy (TTD). Although these diseases were originally associated with defects in DNA repair, transcription deficiencies might be also implicated. By using retinoic acid receptor beta isoform 2 (RARB2) as a model in several cells bearing mutations in genes encoding TFIIH subunits, we observed that (1) the recruitment of the TFIIH complex was altered at the activated RARB2 promoter, (2) TFIIH participated in the recruitment of nucleotide excision repair (NER) factors during transcription in a manner different from that observed during NER, and (3) the different TFIIH variants disturbed transcription by having distinct consequences on post-translational modifications of histones, DNA-break induction, DNA demethylation, and gene-loop formation. The transition from heterochromatin to euchromatin was disrupted depending on the variant, illustrating the fact that TFIIH, by contributing to NER factor recruitment, orchestrates chromatin remodeling. The subtle transcriptional differences found between various TFIIH variants thus participate in the phenotypic variability observed among XP, XP/CS, and TTD individuals. Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  5. Stress-induced changes of hippocampal NMDA receptors: modulation by duloxetine treatment.

    Directory of Open Access Journals (Sweden)

    Francesca Calabrese

    Full Text Available It is now well established that the glutamatergic system contributes to the pathophysiology of depression. Exposure to stress, a major precipitating factor for depression, enhances glutamate release that can contribute to structural abnormalities observed in the brain of depressed subjects. On the other hand, it has been demonstrated that NMDA antagonists, like ketamine, exert an antidepressant effect at preclinical and clinical levels. On these bases, the purpose of our study was to investigate whether chronic mild stress is associated with specific alterations of the NMDA receptor complex, in adult rats, and to establish whether concomitant antidepressant treatment could normalize such deficits. We found that chronic stress increases the expression of the obligatory GluN1 subunit, as well as of the accessory subunits GluN2A and GluN2B at transcriptional and translational levels, particularly in the ventral hippocampus. Concomitant treatment with the antidepressant duloxetine was able to normalize the increase of glutamatergic receptor subunit expression, and correct the changes in receptor phosphorylation produced by stress exposure. Our data suggest that prolonged stress, a condition that has etiologic relevance for depression, may enhance glutamate activity through post-synaptic mechanisms, by regulating NMDA receptors, and that antidepressants may in part normalize such changes. Our results provide support to the notion that antidepressants may exert their activity in the long-term also via modulation of the glutamatergic synapse.

  6. PGE2 receptor EP3 inhibits water reabsorption and contributes to polyuria and kidney injury in a streptozotocin-induced mouse model of diabetes.

    Science.gov (United States)

    Hassouneh, Ramzi; Nasrallah, Rania; Zimpelmann, Joe; Gutsol, Alex; Eckert, David; Ghossein, Jamie; Burns, Kevin D; Hébert, Richard L

    2016-06-01

    The first clinical manifestation of diabetes is polyuria. The prostaglandin E2 (PGE2) receptor EP3 antagonises arginine vasopressin (AVP)-mediated water reabsorption and its expression is increased in the diabetic kidney. The purpose of this work was to study the contribution of EP3 to diabetic polyuria and renal injury. Male Ep 3 (-/-) (also known as Ptger3 (-/-)) mice were treated with streptozotocin (STZ) to generate a mouse model of diabetes and renal function was evaluated after 12 weeks. Isolated collecting ducts (CDs) were microperfused to study the contribution of EP3 to AVP-mediated fluid reabsorption. Ep 3 (-/-)-STZ mice exhibited attenuated polyuria and increased urine osmolality compared with wild-type STZ (WT-STZ) mice, suggesting enhanced water reabsorption. Compared with WT-STZ mice, Ep 3 (-/-)-STZ mice also had increased protein expression of aquaporin-1, aquaporin-2, and urea transporter A1, and reduced urinary AVP excretion, but increased medullary V2 receptors. In vitro microperfusion studies indicated that Ep 3 (-/-) and WT-STZ CDs responded to AVP stimulation similarly to those of wild-type mice, with a 60% increase in fluid reabsorption. In WT non-injected and WT-STZ mice, EP3 activation with sulprostone (PGE2 analogue) abrogated AVP-mediated water reabsorption; this effect was absent in mice lacking EP3. A major finding of this work is that Ep 3 (-/-)-STZ mice showed blunted renal cyclooxygenase-2 protein expression, reduced renal hypertrophy, reduced hyperfiltration and reduced albuminuria, as well as diminished tubular dilation and nuclear cysts. Taken together, the data suggest that EP3 contributes to diabetic polyuria by inhibiting expression of aquaporins and that it promotes renal injury during diabetes. EP3 may prove to be a promising target for more selective management of diabetic kidney disease.

  7. m-Trifluoromethyl-diphenyl diselenide promotes resilience to social avoidance induced by social defeat stress in mice: Contribution of opioid receptors and MAPKs.

    Science.gov (United States)

    Rosa, Suzan Gonçalves; Pesarico, Ana Paula; Nogueira, Cristina Wayne

    2018-03-02

    Depressive symptoms precipitated by stress are prevalent in population. In experimental models of social stress, endogenous opioids mediate different aspects of defensive and submissive behaviors. The present study investigated the opioid receptors, mitogen-activated protein kinase (MAPKs) and protein kinase B (Akt) contribution to m-trifluoromethyl-diphenyl diselenide [(m-CF 3 -PhSe) 2 ] effects on social avoidance induced by social defeat stress (SDS). Adult Swiss mice were subjected to SDS and treated with (m-CF 3 -PhSe) 2 (5 to 25mg/kg) for 7days. After that, the mice performed locomotor and social avoidance tests. The opioid receptors, MAPKs and Akt protein contents were determined in the prefrontal cortical samples of mice. Firstly, the mice were segregated in susceptible or resilient subpopulation based on their social avoidance induced by stress. (m-CF 3 -PhSe) 2 (25mg/kg) was effective against the stress-induced social avoidance and improved social interaction behavior in mice. SDS increased the μ and κ protein contents but reduced those of δ opioid receptors in susceptible mice. Resilient and (m-CF 3 -PhSe) 2 -treated mice had no alteration in the levels of opioid receptors. Moreover, (m-CF 3 -PhSe) 2 was effective against the increase of c-Jun N-terminal kinase (JNK) and the decrease of Akt phosphorylation protein contents induced by SDS in susceptible mice. The protein content of extracellular signal-regulated kinase (ERK) phosphorylation was reduced in both susceptible and resilient mice, whereas p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation was increased only in resilient mice. (m-CF 3 -PhSe) 2 was partially effective against the pERK decrease and ineffective against the increase in p38 MAPK phosphorylation in mice subjected to SDS. These results suggest that the modulation of protein contents of opioid receptors, JNK and Akt phosphorylation is associated with resilience to SDS promoted by (m-CF 3 -PhSe) 2 in mice. Copyright

  8. Characterization of fimbrial subunits from Bordetella species

    NARCIS (Netherlands)

    Mooi, F.R.; Heide, H.G.J. van der; Avest, A.R. ter; Welinder, K.G.; Livey, I.; Zeijst, B.A.M. van der; Gaastra, W.

    Using antisera raised against serotype 2 and 3 fimbrial subunits from Bordetella pertussis, serologically related polypeptides were detected in Bordetella bronchiseptica, Bordetella parapertussis and Bordetella avium strains. The two B. pertussis fimbrial subunits, and three of the serologically

  9. AMPA/kainate glutamate receptors contribute to inflammation, degeneration and pain related behaviour in inflammatory stages of arthritis

    Science.gov (United States)

    Bonnet, Cleo S; Williams, Anwen S; Gilbert, Sophie J; Harvey, Ann K; Evans, Bronwen A; Mason, Deborah J

    2015-01-01

    Objectives Synovial fluid glutamate concentrations increase in arthritis. Activation of kainate (KA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors (GluRs) increase interleukin-6 (IL-6) release and cause arthritic pain, respectively. We hypothesised that AMPA and KA GluRs are expressed in human arthritis, and that intra-articular NBQX (AMPA/KA GluR antagonist) prevents pain and pathology in antigen-induced arthritis (AIA). Methods GluR immunohistochemistry was related to synovial inflammation and degradation in osteoarthritis (OA) and rheumatoid arthritis (RA). A single intra-articular NBQX injection was given at induction, and knee swelling and gait of AIA and AIA+NBQX rats compared over 21 days, before imaging, RT-qPCR, histology and immunohistochemistry of joints. Effects of NBQX on human primary osteoblast (HOB) activity were determined. Results AMPAR2 and KA1 immunolocalised to remodelling bone, cartilage and synovial cells in human OA and RA, and rat AIA. All arthritic tissues showed degradation and synovial inflammation. NBQX reduced GluR abundance, knee swelling (parthritis. PMID:24130267

  10. Toll-like receptor 4 signaling in neurons of trigeminal ganglion contributes to nociception induced by acute pulpitis in rats.

    Science.gov (United States)

    Lin, Jia-Ji; Du, Yi; Cai, Wen-Ke; Kuang, Rong; Chang, Ting; Zhang, Zhuo; Yang, Yong-Xiang; Sun, Chao; Li, Zhu-Yi; Kuang, Fang

    2015-07-30

    Pain caused by acute pulpitis (AP) is a common symptom in clinical settings. However, its underlying mechanisms have largely remained unknown. Using AP model, we demonstrated that dental injury caused severe pulp inflammation with up-regulated serum IL-1β. Assessment from head-withdrawal reflex thresholds (HWTs) and open-field test demonstrated nociceptive response at 1 day post injury. A consistent up-regulation of Toll-like receptor 4 (TLR4) in the trigeminal ganglion (TG) ipsilateral to the injured pulp was found; and downstream signaling components of TLR4, including MyD88, TRIF and NF-κB, and cytokines such as TNF-α and IL-1β, were also increased. Retrograde labeling indicated that most TLR4 positve neuron in the TG innnervated the pulp and TLR4 immunoreactivity was mainly in the medium and small neurons. Double labeling showed that the TLR4 expressing neurons in the ipsilateral TG were TRPV1 and CGRP positive, but IB4 negative. Furthermore, blocking TLR4 by eritoran (TLR4 antagonist) in TGs of the AP model significantly down-regulated MyD88, TRIF, NF-κB, TNF-α and IL-1β production and behavior of nociceptive response. Our findings suggest that TLR4 signaling in TG cells, particularly the peptidergic TRPV1 neurons, plays a key role in AP-induced nociception, and indicate that TLR4 signaling could be a potential therapeutic target for orofacial pain.

  11. Toll-Like Receptor 4 Activation Contributes to Diabetic Bladder Dysfunction in a Murine Model of Type 1 Diabetes.

    Science.gov (United States)

    Szasz, Theodora; Wenceslau, Camilla F; Burgess, Beth; Nunes, Kenia P; Webb, R Clinton

    2016-12-01

    Diabetic bladder dysfunction (DBD) is a common urological complication of diabetes. Innate immune system activation via Toll-like receptor 4 (TLR4) leads to inflammation and oxidative stress and was implicated in diabetes pathophysiology. We hypothesized that bladder hypertrophy and hypercontractility in DBD is mediated by TLR4 activation. Wild-type (WT) and TLR4 knockout (TLR4KO) mice were made diabetic by streptozotocin (STZ) treatment, and bladder contractile function and TLR4 pathway expression were evaluated. Immunohistochemistry confirmed the expression of TLR4 in human and mouse bladder. Recombinant high-mobility group box protein 1 (HMGB1) increased bladder TLR4 and MyD88 expression and enhanced contractile response to electrical field stimulation. Bladder expression of TLR4 and MyD88 and serum expression of HMGB1 were increased in STZ compared with control mice. Carbachol (CCh)-mediated contraction was increased in bladders from STZ mice, and TLR4 inhibitor CLI-095 attenuated this increase. Induction of diabetes by STZ in WT mice increased bladder weight and contractile responses to CCh and to electrical field stimulation. TLR4KO mice were not protected from STZ-induced diabetes; however, despite levels of hyperglycemia similar to those of WT STZ mice, TLR4KO STZ mice were protected from diabetes-induced bladder hypertrophy and hypercontractility. These data suggest that TLR4 activation during diabetes mediates DBD-associated bladder hypertrophy and hypercontractility. © 2016 by the American Diabetes Association.

  12. A family of photoswitchable NMDA receptors

    Science.gov (United States)

    Berlin, Shai; Szobota, Stephanie; Reiner, Andreas; Carroll, Elizabeth C; Kienzler, Michael A; Guyon, Alice; Xiao, Tong; Trauner, Dirk; Isacoff, Ehud Y

    2016-01-01

    NMDA receptors, which regulate synaptic strength and are implicated in learning and memory, consist of several subtypes with distinct subunit compositions and functional properties. To enable spatiotemporally defined, rapid and reproducible manipulation of function of specific subtypes, we engineered a set of photoswitchable GluN subunits ('LiGluNs'). Photo-agonism of GluN2A or GluN2B elicits an excitatory drive to hippocampal neurons that can be shaped in time to mimic synaptic activation. Photo-agonism of GluN2A at single dendritic spines evokes spine-specific calcium elevation and expansion, the morphological correlate of LTP. Photo-antagonism of GluN2A alone, or in combination with photo-antagonism of GluN1a, reversibly blocks excitatory synaptic currents, prevents the induction of long-term potentiation and prevents spine expansion. In addition, photo-antagonism in vivo disrupts synaptic pruning of developing retino-tectal projections in larval zebrafish. By providing precise and rapidly reversible optical control of NMDA receptor subtypes, LiGluNs should help unravel the contribution of specific NMDA receptors to synaptic transmission, integration and plasticity. DOI: http://dx.doi.org/10.7554/eLife.12040.001 PMID:26929991

  13. Differential regulation of synaptic and extrasynaptic α4 GABA(A) receptor populations by protein kinase A and protein kinase C in cultured cortical neurons.

    Science.gov (United States)

    Bohnsack, John Peyton; Carlson, Stephen L; Morrow, A Leslie

    2016-06-01

    The GABAA α4 subunit exists in two distinct populations of GABAA receptors. Synaptic GABAA α4 receptors are localized at the synapse and mediate phasic inhibitory neurotransmission, while extrasynaptic GABAA receptors are located outside of the synapse and mediate tonic inhibitory transmission. These receptors have distinct pharmacological and biophysical properties that contribute to interest in how these different subtypes are regulated under physiological and pathological states. We utilized subcellular fractionation procedures to separate these populations of receptors in order to investigate their regulation by protein kinases in cortical cultured neurons. Protein kinase A (PKA) activation decreases synaptic α4 expression while protein kinase C (PKC) activation increases α4 subunit expression, and these effects are associated with increased β3 S408/409 or γ2 S327 phosphorylation respectively. In contrast, PKA activation increases extrasynaptic α4 and δ subunit expression, while PKC activation has no effect. Our findings suggest synaptic and extrasynaptic GABAA α4 subunit expression can be modulated by PKA to inform the development of more specific therapeutics for neurological diseases that involve deficits in GABAergic transmission. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Therapeutic potential of Mediator complex subunits in metabolic diseases.

    Science.gov (United States)

    Ranjan, Amol; Ansari, Suraiya A

    2018-01-01

    The multisubunit Mediator is an evolutionary conserved transcriptional coregulatory complex in eukaryotes. It is needed for the transcriptional regulation of gene expression in general as well as in a gene specific manner. Mediator complex subunits interact with different transcription factors as well as components of RNA Pol II transcription initiation complex and in doing so act as a bridge between gene specific transcription factors and general Pol II transcription machinery. Specific interaction of various Mediator subunits with nuclear receptors (NRs) and other transcription factors involved in metabolism has been reported in different studies. Evidences indicate that ligand-activated NRs recruit Mediator complex for RNA Pol II-dependent gene transcription. These NRs have been explored as therapeutic targets in different metabolic diseases; however, they show side-effects as targets due to their overlapping involvement in different signaling pathways. Here we discuss the interaction of various Mediator subunits with transcription factors involved in metabolism and whether specific interaction of these transcription factors with Mediator subunits could be potentially utilized as therapeutic strategy in a variety of metabolic diseases. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  15. Advanced glycation endproduct (AGE) accumulation and AGE receptor (RAGE) up‐regulation contribute to the onset of diabetic cardiomyopathy

    Science.gov (United States)

    Ma, Heng; Li, Shi‐Yan; Xu, Peisheng; Babcock, Sara A.; Dolence, E. Kurt; Brownlee, Michael; Li, Ji

    2008-01-01

    Abstract Diabetic cardiomyopathy is manifested by compromised systolic and diastolic function. This study was designed to examine the role of advanced glycation endproduct (AGE) and AGE receptor (RAGE) in diabetic cardiomyopathy. Heart function was assessed in isolated control and streptozotocin‐induced diabetic hearts following in vivo RAGE gene knockdown using RNA interference. Cardiomyocyte mechanical properties were evaluated including peak shortening (PS), time‐to‐PS (TPS) and time‐to‐90% relengthening (TR90). RAGE was assayed by RT‐PCR and immunoblot. Diabetes significantly enhanced cardiac MG, AGE and RAGE levels accompanied with colocalization of AGE and RAGE in cardiomyocytes. Diabetes‐elicited increase in RAGE was inhibited by in vivo siRNA interference. The AGE formation inhibitor benfotiamine significantly attenuated diabetes‐induced elevation in MG, AGE, RAGE and collagen cross‐linking without affecting hypertriglyceridaemia and hypercholesterolaemia in diabetes. Diabetes markedly decreased LV contractility, as evidenced by reduced ±dP/dt and LV developed pressure (LVDP), which were protected by RAGE gene knockdown. In addition, MG‐derived AGE (MG‐AGE) up‐regulated cardiac RAGE mRNA and triggered cardiomyocyte contractile dysfunction reminiscent of diabetic cardiomyopathy. The MG‐AGE‐elicited prolongation of TPS and TR90 was ablated by an anti‐RAGE antibody in cardiomyocytes. Interestingly, MG‐AGE‐induced cardiomyocyte dysfunction was associated with mitochondrial membrane potential (MMP) depolarization and reduced GSK‐3β inactivation in control cardiomyocytes, similar to those from in vivo diabetes. Treatment with siRNA‐RAGE ablated diabetes‐induced MMP depolarization and GSK‐3β inactivation. Collectively, our result implicated a role of AGE‐RAGE in the pathogenesis of diabetic cardiomyopathy. PMID:19602045

  16. Contribution of scintigraphy for somatostatin receptors in case of non-iodine-fixating metastases of thyroid epithelioma

    International Nuclear Information System (INIS)

    Couty, H.; Andrieu, J.M.; Baudet, L.; Faurous, P.; Artus, J.C.; Jaffiol, C.

    1997-01-01

    The objective of this work was to evaluate the sensitivity and importance of scintigraphy of somatostatin receptors (SSR) in the detection of non iodine-fixating metastases of thyroid epithelioma. We have studied retrospectively 7 patients with a detectable thyroglobulin under hormonal hindrance, amounting after cessation of treatment up to 72 - 1500 μ g/l and a normal post-irradiation therapy (3.7 GBq of 131 I) scintigraphy. These patients (4 F / 3 M), aged from 27 to 74 years (average age, 54 years), were treated by total and ganglionic curettage for papillary cancer of thyroid. Their evolutive surveillance extended from 1 to 24 years (on average, 8.4 years) and they received curative cumulated 131 I activities of 13 to 26 GBq. Each of them benefited by an osseous scintigraphy, of a cervico-thoraco-abdominal TDM and of a 111 In Pentetreotide SSR. The SSR was positive for 4/7 patients showing cervico-mediastinal fixations, the localizations being not detected by TDM. The osseous scintigraphy revealed once to be positive showing a costal lesion already detected by SSR. Cervical non-palpable adenopathies were detected by TDM for one patient. The anatomical-pathological analysis confirmed their thyroid origin. For other two patients the TDM showed suspect pulmonary nodules. These cervical and pulmonary localization were not detected by SSR. The SSR + TDM couple was positive for 6/7 patients. In conclusion, in this series the sensitivity of SSR was estimated to be 57%. Besides, the results obtained showed a net complementarity between the detection performances of SSR and TDM

  17. Toll-like receptor 4 upregulation by angiotensin II contributes to hypertension and vascular dysfunction through reactive oxygen species production.

    Directory of Open Access Journals (Sweden)

    Priscila R De Batista

    Full Text Available Hypertension is considered as a low-grade inflammatory disease, with adaptive immunity being an i