FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2153497461> ?p ?o ?g. }

• W2153497461 endingPage "3729" @default.
• W2153497461 startingPage "3717" @default.
• W2153497461 abstract "Article15 October 2009free access Involvement of NMDAR2A tyrosine phosphorylation in depression-related behaviour Sachiko Taniguchi Sachiko Taniguchi Division of Oncology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Takanobu Nakazawa Corresponding Author Takanobu Nakazawa Division of Oncology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Asami Tanimura Asami Tanimura Department of Neurophysiology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan Search for more papers by this author Yuji Kiyama Yuji Kiyama Division of Neuronal Network, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Tohru Tezuka Tohru Tezuka Division of Oncology, Institute of Medical Science, University of Tokyo, Tokyo, JapanPresent address: Division of Genetics, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan Search for more papers by this author Ayako M Watabe Ayako M Watabe Division of Neuronal Network, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Norikazu Katayama Norikazu Katayama Division of Neuronal Network, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Kazumasa Yokoyama Kazumasa Yokoyama Division of Oncology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Takeshi Inoue Takeshi Inoue Division of Oncology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Hiroko Izumi-Nakaseko Hiroko Izumi-Nakaseko Division of Neuronal Network, Institute of Medical Science, University of Tokyo, Tokyo, JapanPresent address: Department of Pharmacology, School of Medicine, Faculty of Medicine, Toho University, Tokyo, 143-8540, Japan Search for more papers by this author Shigeru Kakuta Shigeru Kakuta Center for Experimental Medicine and Systems Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Katsuko Sudo Katsuko Sudo Center for Experimental Medicine and Systems Biology, Institute of Medical Science, University of Tokyo, Tokyo, JapanPresent address: Animal Research Center, Tokyo Medical University, Tokyo, 160-8402, Japan Search for more papers by this author Yoichiro Iwakura Yoichiro Iwakura Center for Experimental Medicine and Systems Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Hisashi Umemori Hisashi Umemori Molecular & Behavioral Neuroscience Institute and Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Takafumi Inoue Takafumi Inoue Department of Life Science and Bio-science, Faculty of Science and Engineering, Waseda University, Tokyo, Japan Search for more papers by this author Niall P Murphy Niall P Murphy Hatos Center for Neuropharmacology, Department of Psychiatry and Biobehavioral Sciences, UCLA Semel Institute for Neuroscience and Human Behavior, Los Angeles, CA, USA Search for more papers by this author Kouichi Hashimoto Kouichi Hashimoto Department of Neurophysiology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Kawaguchi, Japan Search for more papers by this author Masanobu Kano Masanobu Kano Department of Neurophysiology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan Search for more papers by this author Toshiya Manabe Toshiya Manabe Division of Neuronal Network, Institute of Medical Science, University of Tokyo, Tokyo, Japan Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Kawaguchi, Japan Search for more papers by this author Tadashi Yamamoto Tadashi Yamamoto Division of Oncology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Laboratory of Molecular Biology, NCI, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Sachiko Taniguchi Sachiko Taniguchi Division of Oncology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Takanobu Nakazawa Corresponding Author Takanobu Nakazawa Division of Oncology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Asami Tanimura Asami Tanimura Department of Neurophysiology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan Search for more papers by this author Yuji Kiyama Yuji Kiyama Division of Neuronal Network, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Tohru Tezuka Tohru Tezuka Division of Oncology, Institute of Medical Science, University of Tokyo, Tokyo, JapanPresent address: Division of Genetics, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan Search for more papers by this author Ayako M Watabe Ayako M Watabe Division of Neuronal Network, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Norikazu Katayama Norikazu Katayama Division of Neuronal Network, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Kazumasa Yokoyama Kazumasa Yokoyama Division of Oncology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Takeshi Inoue Takeshi Inoue Division of Oncology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Hiroko Izumi-Nakaseko Hiroko Izumi-Nakaseko Division of Neuronal Network, Institute of Medical Science, University of Tokyo, Tokyo, JapanPresent address: Department of Pharmacology, School of Medicine, Faculty of Medicine, Toho University, Tokyo, 143-8540, Japan Search for more papers by this author Shigeru Kakuta Shigeru Kakuta Center for Experimental Medicine and Systems Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Katsuko Sudo Katsuko Sudo Center for Experimental Medicine and Systems Biology, Institute of Medical Science, University of Tokyo, Tokyo, JapanPresent address: Animal Research Center, Tokyo Medical University, Tokyo, 160-8402, Japan Search for more papers by this author Yoichiro Iwakura Yoichiro Iwakura Center for Experimental Medicine and Systems Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Hisashi Umemori Hisashi Umemori Molecular & Behavioral Neuroscience Institute and Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Takafumi Inoue Takafumi Inoue Department of Life Science and Bio-science, Faculty of Science and Engineering, Waseda University, Tokyo, Japan Search for more papers by this author Niall P Murphy Niall P Murphy Hatos Center for Neuropharmacology, Department of Psychiatry and Biobehavioral Sciences, UCLA Semel Institute for Neuroscience and Human Behavior, Los Angeles, CA, USA Search for more papers by this author Kouichi Hashimoto Kouichi Hashimoto Department of Neurophysiology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Kawaguchi, Japan Search for more papers by this author Masanobu Kano Masanobu Kano Department of Neurophysiology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan Search for more papers by this author Toshiya Manabe Toshiya Manabe Division of Neuronal Network, Institute of Medical Science, University of Tokyo, Tokyo, Japan Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Kawaguchi, Japan Search for more papers by this author Tadashi Yamamoto Tadashi Yamamoto Division of Oncology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Laboratory of Molecular Biology, NCI, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Author Information Sachiko Taniguchi1,‡, Takanobu Nakazawa 1,‡, Asami Tanimura2,‡, Yuji Kiyama3, Tohru Tezuka1, Ayako M Watabe3, Norikazu Katayama3, Kazumasa Yokoyama1, Takeshi Inoue1, Hiroko Izumi-Nakaseko3, Shigeru Kakuta4, Katsuko Sudo4, Yoichiro Iwakura4, Hisashi Umemori5, Takafumi Inoue6, Niall P Murphy7, Kouichi Hashimoto2,8, Masanobu Kano2, Toshiya Manabe3,8 and Tadashi Yamamoto1,9 1Division of Oncology, Institute of Medical Science, University of Tokyo, Tokyo, Japan 2Department of Neurophysiology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan 3Division of Neuronal Network, Institute of Medical Science, University of Tokyo, Tokyo, Japan 4Center for Experimental Medicine and Systems Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan 5Molecular & Behavioral Neuroscience Institute and Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI, USA 6Department of Life Science and Bio-science, Faculty of Science and Engineering, Waseda University, Tokyo, Japan 7Hatos Center for Neuropharmacology, Department of Psychiatry and Biobehavioral Sciences, UCLA Semel Institute for Neuroscience and Human Behavior, Los Angeles, CA, USA 8Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Kawaguchi, Japan 9Laboratory of Molecular Biology, NCI, National Institutes of Health, Bethesda, MD, USA ‡These authors contributed equally to this work *Corresponding author. Division of Oncology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Tel.: +81 3 5449 5305; Fax: +81 3 5449 5413; E-mail: [email protected] The EMBO Journal (2009)28:3717-3729https://doi.org/10.1038/emboj.2009.300 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Major depressive and bipolar disorders are serious illnesses that affect millions of people. Growing evidence implicates glutamate signalling in depression, though the molecular mechanism by which glutamate signalling regulates depression-related behaviour remains unknown. In this study, we provide evidence suggesting that tyrosine phosphorylation of the NMDA receptor, an ionotropic glutamate receptor, contributes to depression-related behaviour. The NR2A subunit of the NMDA receptor is tyrosine-phosphorylated, with Tyr 1325 as its one of the major phosphorylation site. We have generated mice expressing mutant NR2A with a Tyr-1325-Phe mutation to prevent the phosphorylation of this site in vivo. The homozygous knock-in mice show antidepressant-like behaviour in the tail suspension test and in the forced swim test. In the striatum of the knock-in mice, DARPP-32 phosphorylation at Thr 34, which is important for the regulation of depression-related behaviour, is increased. We also show that the Tyr 1325 phosphorylation site is required for Src-induced potentiation of the NMDA receptor channel in the striatum. These data argue that Tyr 1325 phosphorylation regulates NMDA receptor channel properties and the NMDA receptor-mediated downstream signalling to modulate depression-related behaviour. Introduction Depression is a severe neuropsychiatric disorder that features a combination of depressed mood feelings, such as sadness, hopelessness, helplessness, and/or worthlessness. The lifetime risk for major depressive disorder and bipolar disorders in the United States is ∼10% as defined by DSM IV. Challenges of establishing the molecular mechanisms underlying depression and of discovering improved therapeutic agents to treat depression are important (Zarate et al, 2006). The serotonergic, noradrenergic, and dopaminergic systems have received great attention in studies related to mood disorders (Sanacora et al, 2008). In addition to these systems, the glutamatergic pathway, a major mediator of excitatory synaptic transmission in the mammalian brain, has been the focus of the pathophysiological study of mood disorders (Orrego and Villanueva, 1993; Sanacora et al, 2008). The agents that act on the glutamatergic pathway are promising candidates for the treatment of mood disorders (Sanacora et al, 2008). In rodents, treatment with competitive N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptor (NMDA receptor) antagonists produced antidepressant-like effects in several behavioural tasks, including the forced swim test (Trullas and Skolnick, 1990). Genetic evidence for the involvement of the glutamatergic pathway in emotional regulation has been obtained by analysing mice with genetically engineered NMDA receptor genes (Mohn et al, 1999; Miyamoto et al, 2001; Boyce-Rustay and Holmes, 2006). Although these previous findings suggest the involvement of the NMDA receptor in depression-related behaviour, the underlying molecular mechanism remains unclear. A number of neuronal functions such as synaptic plasticity are rapidly and reversibly regulated in response to external factors. Phosphorylation reaction is a key process in the regulation of various neuronal functions because it can rapidly and reversibly change the function of cellular proteins, including NMDA receptor subunits. The NMDA receptor is crucial for development, synaptic plasticity, and neuronal excitotoxicity (Choi, 1988; McDonald and Johnston, 1990; Collingridge and Bliss, 1995). The NMDA receptor is composed of NR1 (GluRζ) and NR2 (GluRε) subunits: the NR1 subunit is essential for the function of NMDAR channels, whereas NR2 subunits (NR2A (GluRε1), NR2B (GluRε2), NR2C (GluRε3), and NR2D (GluRε4)) determine the characteristics of NMDAR channels by forming different heteromeric configurations with the NR1 subunit (Kutsuwada et al, 1992; Monyer et al, 1992; Ishii et al, 1993; Cull-Candy et al, 2001). The NR1 and NR2 subunits contain phosphorylation sites that seem to have key roles in regulating NMDA receptor localization and channel activity, as well as downstream signalling via NMDA receptor-associated proteins (Kornau et al, 1997; Husi et al, 2000; Scannevin and Huganir, 2000; Nakazawa et al, 2001, 2006; Sheng and Kim, 2002; Salter and Kalia, 2004; Chen and Roche, 2007; Okabe, 2007). Biochemical studies have found that non-receptor tyrosine kinases, Src and Fyn, phosphorylate NR2A and NR2B (Yang and Leonard, 2001; Salter and Kalia, 2004; Nakazawa et al, 2001, 2006; Chen and Roche, 2007). NR2A and NR2B contain several tyrosine phosphorylation sites (Yang and Leonard, 2001; Salter and Kalia, 2004; Nakazawa et al, 2001, 2006; Chen and Roche, 2007). Tyr 1472 phosphorylation, the principal tyrosine phosphorylation event on NR2B, regulates amygdaloid synaptic plasticity, and fear-related learning (Nakazawa et al, 2006), however, the role of Tyr 1325 phosphorylation on NR2A remains unknown. In this study, we investigated the role of Tyr 1325 phosphorylation on NR2A with knock-in mice in which Tyr 1325 of NR2A is mutated to phenylalanine. Using biochemical, genetic, and electrophysiological approaches with the knock-in mice, we provide evidence of a critical involvement of Tyr 1325 phosphorylation in depression-related behaviour. Results Tyr 1325 as one of the principal Src family kinase-mediated phosphorylation sites The NR2A subunit of the NMDA receptor contains 25 tyrosine residues in the intracellular C-terminal region. To determine the Src family kinase-mediated NR2A phosphorylation sites, we constructed GST fusion proteins containing truncated segments of the NR2A C-terminal region (termed GST–C1, GST–C2, and GST–C3) (Figure 1A). These fusion proteins were phosphorylated by baculovirally expressed Fyn in the presence of [γ-32P]ATP. The phosphorylated proteins were subjected to tryptic phosphopeptide mapping. As shown in Figure 1B, highly phosphorylated peptides P1–P7 were generated from the phosphorylated GST–fusion proteins. To identify the tyrosine residue in most prominently phosphorylated P7 peptide, we constructed Y1325F (conversion of Tyr 1325 to Phe 1325)–GST–C2 protein by site-directed mutagenesis, and performed the in vitro phosphorylation assay as above. Conversion of Tyr 1325 to Phe 1325 resulted in generation of a tryptic phosphopeptide map that lacked phosphopeptide P7, suggesting that Tyr 1325 was most prominently phosphorylated by Fyn in vitro (Figure 1B). Similarly, phosphorylated tyrosines in other phosphopeptides were examined by introducing YF mutations in the GST–C1 and GST–C2 proteins. We found that Tyr 943, Tyr 1105, Tyr 1118, Tyr 1187, Tyr 1246, and Tyr 1267 were also phosphorylated in vitro (data not shown). As Tyr 1325 was most strongly phosphorylated in vitro, we examined whether Tyr 1325 was phosphorylated in cells (Figure 1C). HEK 293T cells were transfected with expression plasmids encoding wild-type or Y1325F mutant forms of the NR2A subunit along with a plasmid encoding Src YF, a constitutively active form of Src. Immunoblotting of NR2A immunoprecipitates with the 4G10 anti-phosphotyrosine antibody revealed that Y1325F mutation significantly decreased the tyrosine phosphorylation level of the NR2A subunits (n=4; *P<0.001, Student's t test; Figure 1C). The data suggest that Tyr 1325 was one of the principal Src-mediated phosphorylation sites in HEK 293T cells as well as in vitro. Figure 1.Identification of Tyr 1325 as one of the principal Src family kinase-mediated phosphorylation sites of the NR2A subunit. (A) Schematic diagram of GST fusion proteins containing the intracellular C-terminal region of the NR2A subunit. (B) Two-dimensional tryptic phosphopeptide maps of GST–C1, GST–C2, GST–C2–Y1325F, and GST–C3. The dot in each map shows the origin of electrophoresis. Note that P7 (Tyr 1325) was most prominently phosphorylated in vitro. (C) Identification of Tyr 1325 as one of the principal phosphorylation sites in HEK 293T cells. HEK293T cells were transfected with combinations of expression plasmids for the NR2A subunit, NR2A Y1325F mutants, and active Src (SrcYF) or inactive Src (SrcKM). NR2A immunoprecipitates from cell lysates were subjected to immunoblotting using the anti-pY antibody (4G10) (top), followed by a re-blot using the anti-NR2A antibody (middle). The expression levels of Src were confirmed by immunoblotting (bottom). *P<0.001, n=4, Student's t-test. Note that all lanes are from the same gel, but lanes originally present between lanes 2 and 3 have been omitted for brevity. Download figure Download PowerPoint Point mutation of Tyr 1325 on NR2A blocks Src-mediated potentiation of NMDA receptor currents We next addressed the biological significance of Tyr 1325 phosphorylation. As NR2A phosphorylation by Src is suggested to potentiate NMDA receptor currents (Wang and Salter, 1994; Kohr and Seeburg, 1996; Yu et al, 1997), we examined the effect of null phosphorylation at Tyr 1325 by introducing Y1325F mutation in NR2A. Heteromeric NMDA receptor channels consisting of NR1 and wild-type NR2A or Y1325F mutant (Y1325F–NR2A) were transiently expressed in HEK 293T cells and NMDA-evoked whole cell currents were recorded. The current mediated by the NMDA receptor channel with wild-type NR2A subunits was potentiated by the application of the Src protein through patch pipette (Figure 2A). In contrast, the current mediated by the NMDA receptor channel with Y1325F–NR2A subunits was minimally potentiated by the Src protein (Figure 2A). To confirm the lack of Src-dependent NMDA receptor potentiation by Y1325F mutation, we examined the effect on Ca2+ influx. HEK293T cells were transfected with expression plasmids encoding NR1 and wild-type (WT) or Y1325F mutant forms of NR2A together with or without active Src. After that the cells were loaded with fura2/AM and stimulated using NMDA. As shown in Figure 2B, a marked increase of Ca2+ concentration was observed in cells expressing NR1/WT–NR2A and active Src after NMDA stimulation, as compared with that in cells expressing NR1/WT–NR2A alone. In contrast, the level of increase in Ca2+ in cells expressing NR1/Y1325F–NR2A and active Src was virtually identical to that in cells expressing NR1/Y1325F–NR2A alone (Figure 2B). These data suggest that Tyr 1325 phosphorylation is required for Src-mediated potentiation of NMDA receptor currents to modulate Ca2+ signalling downstream of NMDA receptors. Figure 2.Point mutation at Tyr 1325 on the NR2A subunit blocks Src-mediated potentiation of recombinant NMDA receptor currents in HEK 293T cells. (A) Application of Src differentially potentiated NMDA-activated currents mediated by recombinant NR1–wild-type (WT) NR2A channels (upper), but not those mediated by recombinant NR1–Y1325F NR2A channels (lower). Peak currents evoked by NMDA applications (100 μM NMDA and 10 μM glycine: 0.5 s) at 30-s intervals were normalized to the first responses at 3 min after establishing whole-cell configuration (time: 0 min; holding potential: −60 mV). Src (30 U/ml) was added to the internal pipette solution. WT NR2A (control), n=5; WT NR2A (+Src), n=6; Y1325F NR2A (control), n=4; Y1325F NR2A (+Src), n=5. Representative averaged whole-cell currents evoked at −60 mV by the NMDA application are shown in the left panel and the traces recorded at 3 min (grey) and 20 min (black) are superimposed. Scale bars: 0.2 s; 100 pA. (B) Src-dependent increment of free calcium levels in cells expressing NR1/wild-type NR2A, but not NR1/Y1325F–NR2A channel. Intracellular free calcium levels, before and after NMDA stimulation in fura-2/AM-loaded cells, were monitored by spectrofluorometry. Increase in the ratio of the fluorescence intensities at excitation wavelengths of 340 nm (Fex340) and 380 nm (Fex380) after NMDA stimulation are shown (n=4, *P<0.05, Student's t-test). NS: not significant. Download figure Download PowerPoint Generation of NR2A Y1325F knock-in mice To examine the physiological role of NR2A Tyr 1325 phosphorylation, we generated mutant mice in which Tyr 1325 was substituted with Phe 1325 by a knock-in technique (Figure 3A). The success of these procedures was confirmed by Southern blot and PCR analysis (data not shown). We confirmed the absence of Tyr 1325 phosphorylation in homozygous knock-in mice (YF/YF mice) by immunoblot analysis using the anti-pY1325 antibody that specifically recognizes NR2A phosphorylated at Tyr 1325 (Figure 3B and Supplementary Figure 1). The expression level of NR2A in YF/YF mice was virtually identical to that in wild-type littermates (WT/WT mice) (Figure 3B). In YF/YF mice, the level of NR2A tyrosine phosphorylation was significantly reduced as compared with that in WT/WT mice (n=3; *P<0.001, Student's t test; Figure 3C), indicating that Tyr 1325 was one the major phosphorylation sites in vivo as well as in vitro (Figure 1). YF/YF mice were born according to Mendelian genetics and appeared healthy (data not shown). Histological analysis with Nissl-stained coronal sections of central nervous system structures from YF/YF mice did not show any gross abnormalities in cytoarchitecture (Figure 3D). These findings suggest that Tyr 1325 phosphorylation may be relevant to the NMDAR function in mature brain. Figure 3.Generation of mice with a mutation of Tyr 1325 phosphorylation site on NR2A. (A) Schematic representations of the structures of wild-type, targeting vector, and targeted and Neo-targeted NR2A alleles. TK, thymidine kinase gene; neo, neomycin resistance gene. (B) Absence of Tyr 1325 phosphorylation in homozygous YF/YF mice. Equal amounts of brain lysates from WT/WT and YF/YF mice were probed with the anti-pY1325 antibody followed by a re-blot using the anti-NR2A antibody. (C) NR2A tyrosine-phosphorylation in YF/YF mice. Equal amounts of NR2A immunoprecipitates from striatal lysates of WT/WT and YF/YF mice were probed with the anti-phosphotyrosine (4G10) antibody followed by a re-blot with the anti-NR2A antibody. A representative blot is shown on the left (*P<0.01, n=3, Student's t-test). (D) Nissl-stained coronal sections of central nervous system structures including striatum from WT/WT and YF/YF mice. Cpu: Caudata-putamen. Download figure Download PowerPoint Impaired Src-induced potentiation of NMDA receptor-mediated excitatory postsynaptic currents in medium spiny neurons in acute slices from YF/YF mice To examine the effect of Tyr 1325 phosphorylation on NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) in vivo, we performed electrophysiological experiments on medium spiny neurons. Two glass micropipettes were placed in the cerebral cortex or in the underlying white matter to stimulate the corticostriatal axons. Stimulus pulses (duration: 0.1 ms; intensity: 0–80 V) were then applied between the pipettes to evoke EPSCs in medium spiny neurons. Application of the Src protein through a patch pipette potentiated the NMDA receptor-mediated EPSCs in WT/WT slices (Figure 4). In contrast, NMDA receptor-mediated EPSCs were not significantly changed by the Src protein in YF/YF slices (Figure 4). These results suggest that Tyr 1325 phosphorylation is required for Src-induced potentiation of the NMDA receptor-mediated EPSCs in medium spiny neurons, which is consistent with findings from recombinantly expressed NMDA receptor in HEK 293T cells (Figure 2). Figure 4.Src-induced potentiation of NMDA receptor-mediated EPSCs is abolished in medium spiny neurons in acute striatal slices from YF/YF mice. (A) Examples of NMDA receptor-mediated EPSCs. (B) Src-dependent change in the peak amplitude of NMDA receptor-mediated EPSCs in medium spiny neurons from WT/WT and YF/YF mice. WT/WT, n=15; YF/YF, n=11; *P=0.036, paired t-test. Download figure Download PowerPoint Antidepressant-like phenotypes in the forced swim test and the tail suspension test in YF/YF mice We performed a battery of behavioural tests to examine sensory and motor functions as well as cognition and anxiety of YF/YF mice. The tests revealed that YF/YF mice were less immobile than WT/WT mice in the tail suspension test (Figure 5A and B), one of the most widely used models for assessing antidepressant-like activity in mice (Cryan et al, 2005). YF/YF mice also showed significantly less immobility than WT/WT mice in the forced swim test (Figure 5C and D), another test for assessing antidepressant-like activity (Petit-Demouliere et al, 2005). Given that, the higher the mobility, mice are less depressive (Cryan et al, 2005; Petit-Demouliere et al, 2005), we postulated that YF/YF mice were less susceptible to depression than WT/WT mice. Alternatively, the reduced immobility of YF/YF mice in these tests might have been caused by the increase in spontaneous activity (Crawley, 2007). To check this possibility, we performed the open field test to measure spontaneous locomotor activity of YF/YF mice (Figure 5E). The results showed that there were no significant differences between WT/WT and YF/YF mice in the number of rearing (measure the number of beam breaks) (WT/WT, 47.9±4.10, n=13; YF/YF, 51.5±4.49, n=13; F(1,24)=0.35, P=0.56, one-way ANOVA) and the total distance travelled during the test (WT/WT, 3435±191 cm, n=13; YF/YF, 3400±182 cm, n=13; F(1,24)=0.018, P=0.89, one-way ANOVA), suggesting that spontaneous activity was virtually normal in YF/YF mice. As alteration in fearfulness can also affect the performance in the tests for depression (Crawley, 2007), we also analysed the anxiety-related behaviour. We did not detect any significant abnormality in YF/YF mice in the open field test (the time spent in the centre, WT/WT, 13.0±1.78%, n=13; YF/YF, 10.2±1.10%, n=13; F(1,24)=1.83, P=0.19, one-way ANOVA; Figure 5E), the light–dark emergence test (the time spent in the lighted area: WT/WT, 40.9±3.15%, n=13; YF/YF, 42.6±4.14%, n=13; F(1,24)=0.11, P=0.74, one-way ANOVA; the number of transitions: WT/WT, 24.9±2.10, n=13; YF/YF, 21.7±2.96, n=13; F(1,24)=0.79, P=0.38, one-way ANOVA; Figure 5F), the social interaction test (the number of social interactions: WT/WT, 14.5±1.7, n=11; YF/YF, 14.5±0.8, n=11; F(1,20)=0.002, P=0.96, one-way ANOVA; the social interaction time, WT/WT, 12.7±2.06%, n=11; YF/YF, 16.4±2.66, n=11; F(1,20)=1.18, P=0.28, one-way ANOVA; Figure 5G), and the elevated plus maze test in YF/YF mice (the time on open arms: WT/WT, 15.68±1.48%, n=13; YF/YF, 16.45±2.85%, n=13; F(1,24)=0.06, P=0.81, one-way ANOVA; the number of entries into open arms: WT/WT, 28.2±2.02, n=13; YF/YF, 30.7±2.76, n=13; F(1,24)=0.55, P=0.47, one-way ANOVA; the number of head dips: WT/WT, 42.5±7.69, n=10; YF/YF, 43.5±9.89, n=10; F(1,18)=0.006, P=0.94, one-way ANOVA; Figure 5H). In addition, the performance of YF/YF mice in the Morris water maze test, contextual fear conditioning test, auditory fear conditioning test, acoustic startle response test, pre-pulse inhibition test, tail flick test, and hot plate test appeared normal (Supplementary Figure 2-4). These results suggest that YF/YF mice showed reduced susceptibility to depression, but other general behaviours, such as locomotor activity, cognitive function, anxiety-related behaviour, startle response, and pain behaviour are normal. Figure 5.Reduced depression-related behaviour in YF/YF mice. (A) Percent immobility of WT/WT and YF/YF mice in the tail suspension test. (B) Summary of immobility in the tail suspension test during the" @default.
• W2153497461 created "2016-06-24" @default.
• W2153497461 creator A5013307467 @default.
• W2153497461 creator A5020655908 @default.
• W2153497461 creator A5022594173 @default.
• W2153497461 creator A5024136589 @default.
• W2153497461 creator A5031800699 @default.
• W2153497461 creator A5031862986 @default.
• W2153497461 creator A5032763504 @default.
• W2153497461 creator A5034430377 @default.
• W2153497461 creator A5044756754 @default.
• W2153497461 creator A5046803418 @default.
• W2153497461 creator A5053873787 @default.
• W2153497461 creator A5057500761 @default.
• W2153497461 creator A5058411389 @default.
• W2153497461 creator A5059569764 @default.
• W2153497461 creator A5060740378 @default.
• W2153497461 creator A5062513710 @default.
• W2153497461 creator A5072607053 @default.
• W2153497461 creator A5073398296 @default.
• W2153497461 creator A5074919264 @default.
• W2153497461 creator A5087333755 @default.
• W2153497461 date "2009-10-15" @default.
• W2153497461 modified "2023-10-13" @default.
• W2153497461 title "Involvement of NMDAR2A tyrosine phosphorylation in depression-related behaviour" @default.
• W2153497461 cites W1500368251 @default.
• W2153497461 cites W1506555898 @default.
• W2153497461 cites W1563427807 @default.
• W2153497461 cites W1594111906 @default.
• W2153497461 cites W1970851711 @default.
• W2153497461 cites W1978265841 @default.
• W2153497461 cites W1978329946 @default.
• W2153497461 cites W1979406476 @default.
• W2153497461 cites W1983188620 @default.
• W2153497461 cites W1984057909 @default.
• W2153497461 cites W1990625157 @default.
• W2153497461 cites W1991449336 @default.
• W2153497461 cites W1996166611 @default.
• W2153497461 cites W2011480808 @default.
• W2153497461 cites W2016425872 @default.
• W2153497461 cites W2018828094 @default.
• W2153497461 cites W2023979631 @default.
• W2153497461 cites W2024036641 @default.
• W2153497461 cites W2024431713 @default.
• W2153497461 cites W2024624930 @default.
• W2153497461 cites W2024945446 @default.
• W2153497461 cites W2028135910 @default.
• W2153497461 cites W2030590865 @default.
• W2153497461 cites W2030756376 @default.
• W2153497461 cites W2034424952 @default.
• W2153497461 cites W2035147381 @default.
• W2153497461 cites W2035856577 @default.
• W2153497461 cites W2037111632 @default.
• W2153497461 cites W2051455839 @default.
• W2153497461 cites W2053980736 @default.
• W2153497461 cites W2060536994 @default.
• W2153497461 cites W2067261800 @default.
• W2153497461 cites W2072240979 @default.
• W2153497461 cites W2074907255 @default.
• W2153497461 cites W2081108290 @default.
• W2153497461 cites W2086305648 @default.
• W2153497461 cites W2090966488 @default.
• W2153497461 cites W2102591195 @default.
• W2153497461 cites W2116635659 @default.
• W2153497461 cites W2145678443 @default.
• W2153497461 cites W2155181385 @default.
• W2153497461 cites W2159891593 @default.
• W2153497461 cites W2164674870 @default.
• W2153497461 cites W2165909414 @default.
• W2153497461 cites W2169826947 @default.
• W2153497461 cites W4251324165 @default.
• W2153497461 cites W4293060098 @default.
• W2153497461 doi "https://doi.org/10.1038/emboj.2009.300" @default.
• W2153497461 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2790487" @default.
• W2153497461 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19834457" @default.
• W2153497461 hasPublicationYear "2009" @default.
• W2153497461 type Work @default.
• W2153497461 sameAs 2153497461 @default.
• W2153497461 citedByCount "81" @default.
• W2153497461 countsByYear W21534974612012 @default.
• W2153497461 countsByYear W21534974612013 @default.
• W2153497461 countsByYear W21534974612014 @default.
• W2153497461 countsByYear W21534974612015 @default.
• W2153497461 countsByYear W21534974612016 @default.
• W2153497461 countsByYear W21534974612017 @default.
• W2153497461 countsByYear W21534974612018 @default.
• W2153497461 countsByYear W21534974612019 @default.
• W2153497461 countsByYear W21534974612020 @default.
• W2153497461 countsByYear W21534974612021 @default.
• W2153497461 countsByYear W21534974612022 @default.
• W2153497461 countsByYear W21534974612023 @default.
• W2153497461 crossrefType "journal-article" @default.
• W2153497461 hasAuthorship W2153497461A5013307467 @default.
• W2153497461 hasAuthorship W2153497461A5020655908 @default.
• W2153497461 hasAuthorship W2153497461A5022594173 @default.
• W2153497461 hasAuthorship W2153497461A5024136589 @default.
• W2153497461 hasAuthorship W2153497461A5031800699 @default.
• W2153497461 hasAuthorship W2153497461A5031862986 @default.

• next