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  • 97
    Millipore mouse anti psd 95
    Postsynaptic localization of AKAP79/150 at excitatory synapses in hippocampal neurons. A, Developmental regulation of AKAP79/150 colocalization with the MAGUK <t>PSD-95.</t> Rat neonatal hippocampal neurons cultured at low density for the indicated number of days were stained for AKAP150 ( red ) and the postsynaptic density MAGUK PSD-95 ( green ). The small panels are magnifications of dendrites. B , Punctate colocalization of AKAP79/150 with PSD-95 on dendritic spines: proximity of AKAP79/150 puncta to presynaptic terminals stained for synapsin but not inhibitory postsynaptic elements stained for GABA A receptors. Neurons (2–3 weeks old) cultured at medium-high density were stained with anti-rat AKAP150 ( red ) and anti-PSD-95, anti-synapsin, or anti-GABA A receptor (all in green ). Codistribution is seen as yellow-orange in composite images. The red and green arrowheads point to puncta for AKAP150 and PSD-95, respectively, that are not colocalized even in “mature” dendrites. The images shown are representative of multiple neurons imaged in more than three experiments for each condition.
    Mouse Anti Psd 95, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 255 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Antibodies Inc psd 95
    Phosphorylation of <t>PSD-95</t> by Cdk5 decreases the binding of Src to PSD-95. A , The impact of the phosphorylation of the N terminus of PSD-95 by Cdk5 on the binding of PSD-95 to Src was evaluated in transfected HEK 293T cells expressing Y527F Src (active), p35-Cdk5 (active), and either wild-type PSD-95 or the alanine mutant of PSD-95 (T19A, S25A, S35A; AAA PSD-95). Src immunoprecipitated from lysates of transfected cultures was analyzed for the amount of AAA PSD-95 coimmunoprecipitated with Src. Quantification shows that Src binds significantly more to AAA PSD-95 compared with wild-type PSD-95 (786.33 ± 156% of wild-type, p = 0.001; n = 4). B , The impact of the phosphorylation of the N terminus of PSD-95 by Cdk5 on the binding of PSD-95 to Src was evaluated in transfected HEK 293T cells expressing Y527F Src (active), p35-DNk5 (inactive), and either wild-type PSD-95 or the phosphorylation-mimic of PSD-95 (T19D, S25D, S35D; DDD PSD-95). Src immunoprecipitated from lysates of transfected cultures was analyzed for the amount of DDD PSD-95 coimmunoprecipitated with Src. Quantification shows that Src binds less to DDD PSD-95 compared with wild-type PSD-95 (38.28 ± 19.01% of wild-type; p = 0.162; n = 3).
    Psd 95, supplied by Antibodies Inc, used in various techniques. Bioz Stars score: 91/100, based on 241 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson psd 95
    ( A ) Left: Representative immunoblot of membrane-enriched, Triton-X insoluble whole-brain homogenate lysates from young (1.5 mo) and aged ( > 2 years) WT FVB mice, separated on a 4–18% Tris-HCl acrylamide gel, and probed for <t>PSD-95.</t> Right: densitometric quantifications of PSD-95 normalized by GAPDH, expressed as folds of 1.5 mo (***p
    Psd 95, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 109 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    GeneTex psd 95
    (A) Representative Western blots and densitometry analysis of GLT1, <t>PSD-95,</t> and SNAP-25 following 7 days treatment with Aβo, oleocanthal, or combination. Aβo significantly reduced GLT1 and PSD-95 expressions that were rectified by oleocanthal to levels above their baseline expressions; SNAP-25 was not significantly altered by the 7 days exposure to Aβo. (B) Representative Western blots and densitometry analysis of APP, sAPPα, and sAPPβ; 7 days exposure to Aβo significantly increased APP, sAPPα, and sAPPβ levels that were not modulated by oleocanthal. LRP1 was not significantly altered by the treatments. Data is presented as mean ± SD (*P
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    93
    NeuroMab psd 95
    1NM-PP1 suppresses punctate distribution of <t>PSD-95</t> dose-dependently in TrkB F616A neurons. (A) The top row shows representative neurons immunostainted with anti-PSD-95. Typical secondary dendritic branches analyzed are shown in 48 μm 2 rectangles in the middle row. These branches are processed after the thresholding shown in the bottom row. The scales in the top and bottom rows show 10 and 1 μm, respectively. (B) Graph showing averaged PSD-95 intensities in somata. (C) Graph showing quantification of PSD-95 puncta total pixel intensities. Note that all three concentrations of 1NM-PP1 result in reduced total PSD-95 puncta intensity. (D) In this graph, PSD-95 puncta intensities are normalized to somal PSD-95 intensities. In each condition, 16 branches from eight cells (chosen from the two different dissociations) were analyzed. Error bars represent SEM.
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    92
    Santa Cruz Biotechnology psd 95
    Morphine decreases the phosphorylation of GluR1 at Ser 845 and inhibits the interaction between GluR1 and <t>PSD-95</t> through μ-opioid receptor. (A) Neurons were treated with no drug (Ctl) or 10 µM morphine for various durations (3 hours, 6 hours and 1 day) (from left to right). The cell lysates were immunoprecipitated with an anti-GluR1 antibody. The amount of phosphorylated GluR1 at Ser 845 (Phospho-S845; first ), PSD95 proteins ( second ) and total GluR1 subunits ( third ) in the immunoprecipitation complex were detected with appropriate antibodies. Total GluR1 from whole cell extracts (Input) was also determined ( fourth ). (B) The western blots in (A) were quantified with densitometry and normalized to the untreated control ( n = 5). Note that the amount of PSD95 immunoprecipitated by the anti-GluR1 antibody significantly decreases at 6 hours and 1 day after addition of morphine, indicating a dissociation between the postsynaptic density and GluR1 subunits upon drug treatment. (C) Neurons were treated with no drug (Ctl) or 10 µM morphine for 6 hours and 1 day in the absence (vehicle) or presence of 10 µM CTOP. The cell lysates were immunoprecipitated with an anti-GluR1 antibody. The amount of GluR1-S845 phosphorylation (Phospho-S845; first ), PSD95 ( second ) and total GluR1 ( third ) in the immunoprecipitation complex was detected. Total GluR1 subunits from whole cell lysates (Input) were also determined ( fourth ). (D) and ( E ) show densitometric quantifications of western blots in (C) on the amount of phospho-S845 GluR1 and PSD-95 in the receptor complex, respectively (normalized to the untreated control; n = 3). * p
    Psd 95, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 194 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Synaptic Systems psd 95
    Protein levels of glutamatergic markers in the lateral amygdala of human heroin and control subjects from the heroin abuse population (Study II). A. Representative WB images of GluA1 (106 kD), <t>PSD-95</t> (95 kD), mGluR5 (130 kD), Homer 1b/c (45 kD), GluN1 (110) kD and GAPDH (38 kD) in two control and two heroin subjects. B. Comparison of the immunoreactivities between human heroin (n=27–28) and control subjects (n=8–13). Protein levels are presented as percent of mean control values (mean ± SEM). *, p
    Psd 95, supplied by Synaptic Systems, used in various techniques. Bioz Stars score: 92/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Upstate Biotechnology Inc psd 95
    Immunoblots of NR1, NR2A, and actin (a) nNOS, <t>PSD-95,</t> and actin (b) as well as glutamine synthetase (GS), GAD67, and tubulin (c) from 3 representative pairs of controls (Cont) and depressed subjects (Dep) used in the analysis. Each well was loaded with
    Psd 95, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 92/100, based on 203 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    US Biological Life Sciences psd 95
    Subsynaptic localization of dysbindin-1 isoforms. A : Western blotting results on synaptosomal (Syn), synaptic vesicle (SV), presynaptic membrane (PrS), and postsynaptic density (PSD) fractions of the normal HF from three control cases (1–3) in this study. Successful synaptic fractionation was confirmed with synaptophysin as a marker for synaptic vesicles and <t>PSD-95</t> as a marker for the PSD. No selective molecular marker is available for the PrS fraction, including syntaxin-1 since this proves to be ubiquitously distributed on subcellular membranes. β-actin served as the loading control. The blots were probed with PA3111 (1∶1000) for dysbindin-1A and -1C and with UPenn 331 (1∶5000) for dysbindin-1B. In synaptosomes, dysbindin-1A and -1C were found to be predominantly PSD proteins, while dysbindin-1B was found to be predominantly a synaptic vesicle-associated protein. MW = molecular weight marker position. B : Diagram of pre- and/or post-synaptic location of dysbindin-1 isoforms consistent with the Western blotting results just shown and with dysbindin-1 immunohistochemical findings at the electron microscopic level by Talbot et al. [43] .
    Psd 95, supplied by US Biological Life Sciences, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Agilent technologies psd 95
    Knock-down of Tm-agrin in mature hippocampal neurons reduces synapse density Hippocampal neurons fixed at DIV21, 7 days post infection, were immuno-stained for <t>PSD-95</t> (green) to visualize post-synaptic puncta and synaptogamin (red) to label presynaptic boutons. All dendrites were measured and all synapses were counted as described above. Examples of hippocampal neurons from (A) uninfected control, (B) VsiMock-infected and (C) VsiAgrin-infected cultures are shown. Yellow arrowheads indicate examples of overlapping puncta of PSD-95 and synaptotagmin that were counted as synapses. (D) Quantitative assay of synapse density in each group demonstrates that the VsiAgrin-infected neurons had a significantly lower number of synapses per 100μm dendritic length compared to control or VsiMock-infected neurons. Synapse density of VsiMock-infected neurons was not significantly different from uninfected control (N=75 neurons, 3 separate experiments). Bars show the mean ± SEM. Asterisks indicate value significantly different from VsiMock and uninfected control. Bar = 50 μm.
    Psd 95, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Epitomics psd 95
    Caveolin (Cav)-1 knock-out (KO) and Cav-3 KO mice have altered expression and membrane/lipid raft (MLR) localization of key neuronal and glial proteins. Sucrose density fractionation (SDF) and Western blot (WB) of wild-type (WT), Cav-1 KO, and Cav-3 KO brains. Buoyant fractions (BF) contain the cholesterol and sphingolipid enriched MLR, while heavy farctions (HF) contain non-MLR cellular components. (A) WB detection of <t>PSD-95,</t> NR2B, NR1 and TrkB expression in BF and HF. (B) WB detection of toll-like receptor-4 (TLR4), A A2 AR, A 3 AR, and A 1 AR expression in BF and HF. (C) WB detection of LRP-1 and LDL-R expression in BF and HF. Bottom left, WB analysis of GAPDH in whole cell lysates (WCL) from which SDF were generated for each group. Bottom right, WB shows loss of Cav-1 and Cav-3 protein expression in the transgenic mouse used in the present study.
    Psd 95, supplied by Epitomics, used in various techniques. Bioz Stars score: 92/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Trommsdorff psd 95
    Apo ER 2 exon 19 inclusion is lower in human and mouse AD brains compared to non‐ AD samples Schematic of Apo ER 2 expression and signaling via ligand binding (e.g., Reelin, ApoE). Apo ER 2 alternative splicing of exon 19 produces two Apo ER 2 protein isoforms with distinct functions. While both isoforms of Apo ER 2 (+ex19 and Δex19) can bind ligand, a protein domain of Apo ER 2 encoded by exon 19 interacts with <t>PSD</t> 95 to mediate association of Apo ER 2 with the NMDAR complex following Reelin binding. This signaling leads to calcium influx through NMDAR s and the induction of long‐term potentiation ( LTP ). RT – PCR analysis of RNA from mid‐temporal autopsy brain sections obtained from NCI , MCI , or AD subjects. Representative gel of results is shown. Quantitation of RT – PCR products of all samples analyzed from postmortem brain tissues (mean ± s.e.m.). An omnibus significant effect was obtained using the one‐way analysis of variance (one‐way ANOVA ) with Tukey posttest to determine the P ‐value. The number of samples analyzed ( n ) is indicated. RT – PCR analysis of RNA from the hippocampus of 1‐ to 2‐ and 3‐ to 7‐month‐old non‐transgenic ( WT ) or Tg CRND 8 ( AD ) mice. Quantification of the relative ratio of exon 19 inclusion for Tg CRND 8 ( AD ) and WT mice (mean ± s.e.m.; one‐way ANOVA with Tukey posttest). The number of samples analyzed ( n ) is indicated. Source data are available online for this figure.
    Psd 95, supplied by Trommsdorff, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    VANGL2 LTD psd 95
    Punctate and synaptic localisations of Vangl2 with or without PDZ-binding motif. ( a ) Rat hippocampal neurons were transfected with the indicated expression plasmids (upper: GFP-Vangl2 WT, middle: GFP-Vangl2 ΔETSV, lower: mGFP) and cultured for 21 days. GFP fluorescence (green), <t>α-PSD-95</t> immunofluorescence (IF; red) as well as their merged images are presented. Note the punctate localisation of GFP-Vangl2 ΔETSV and the mostly flat signals of mGFP. ( b ) Bar graphs showing the density of the GFP puncta that were formed by the indicated expression constructs along the dendrites. Note the similar levels of puncta formation between GFP-Vangl2 WT and ΔETSV. ( c ) Bar graphs showing the ratio of the PSD-95-associated population of formed GFP puncta for each expression construct. Note that the clusters of Vangl2 ΔETSV are more associated with PSD-95 than those of the simply membrane-tethered protein (mGFP). The highly magnified images of the delimited region are shown in the respective lower panels. Some examples of the co-localised puncta are indicated with arrowheads. The data are presented as mean ± SD. Significant (p
    Psd 95, supplied by VANGL2 LTD, used in various techniques. Bioz Stars score: 93/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Merck & Co psd 95
    Immunoblot analysis of expression ratios of the pre and postsynaptic proteins in Aβ-GFP Tg and non-Tg mice synapse. ( A,B ) Synaptosome fractions from hippocampus (20 μg/lane) were immunoblotted against presynaptic markers ( A ; synaptophysin, VAMP-2, syntaxin-1, and Munc13–1) and postsynaptic markers ( B ; <t>PSD-95,</t> GluN1, GluN2A, GluN2B, GluA2, and neuroligin). We prepared 4 sets of Aβ-GFP Tg/non-Tg samples. Band intensities of Aβ-GFP Tg mice were normalized by those of actin and the expression were calculated ratios with the values of non-Tg mice as 1. There was no difference in expression ratios of the presynaptic proteins we examined. However, expression of the postsynaptic proteins GluN2B, Glu2A, and neuroligin were significantly decreased in Aβ-GFP Tg mice at 3-month-old (*p
    Psd 95, supplied by Merck & Co, used in various techniques. Bioz Stars score: 93/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore psd 95
    Postsynaptic receptor scaffolding proteins and subunits. In contralateral V1, <t>PSD-95</t> ( A ) and gephyrin ( B ) had a similar pattern of changes: fluoxetine alone had no effect, MD alone caused a loss of expression, but combining fluoxetine with MD prevented the MD-induced loss and caused super-compensation above normal levels. GluN1 ( C ) was reduced by fluoxetine regardless of visual experience, whereas MD alone caused an increase. GluA2 ( D ) was unaffected by fluoxetine alone, MD caused an increase, but combing fluoxetine with MD caused a decrease. GluN2B ( E ) was reduced by fluoxetine regardless of visual experience, whereas MD had no effect. GluN2A ( F ) expression of each experimental group was not different from normal animals, but the MDed group had higher expression than either fluoxetine alone or fluoxetine combined with MD. GABAAα3 ( G ) was unaffected by fluoxetine alone, MD caused an increase, but combing fluoxetine with MD prevented the MD-induced increase. GABAAα1 ( H ) was increased by fluoxetine regardless of visual experience, while MD alone had no effect. * p
    Psd 95, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 1112 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Abmart psd 95
    S561A mutation facilitates the interaction between <t>PSD-95</t> and its synaptic binding partners. a , GST pulldown assay for GKAP. GST or GST-SH3-GK WT/S561A beads were incubated with whole-brain lysates. Bound proteins were analyzed by Western blotting with the GKAP antibody. b , quantification of blots in a . The error bar in the scatter plot represents S.D. n = 4. *, p
    Psd 95, supplied by Abmart, used in various techniques. Bioz Stars score: 91/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck KGaA psd 95
    NR1 and <t>PSD-95</t> proteins in PSD-enriched fractions from prefrontal cortex are reduced in people with schizophrenia. NR1 protein levels, normalized to β-III-tubulin, were quantified by western blot images captured by Chemidoc (representative blot captured on film shown in a ). Single bands for NR1 and β-III-tubulin were present at the predicted sizes (~120 and 50 kDa, respectively). NR1 protein levels (average of three replicates) were decreased compared to matched controls ( c ). PSD-95 protein levels, normalized to actin, were quantified by western blot images captured on film (representative blot captured on film shown in b ). Single bands for PSD-95 and actin were present in the predicted sizes (~105 and 42 kDa, respectively). PSD-95 protein levels (average of two replicates) were decreased compared with matched controls ( d ). ** P
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    93
    Thermo Fisher psd 95
    <t>PSD-95</t> deletion impairs retrieval and stability of remote fear memories ( a ) Schematic of experimental design for testing remote retrieval of a cued fear memory. ( b ) PSD-95 GK mice froze less than WT controls during remote fear memory retrieval (n=8–9). ( c ) Schematic of experimental design for testing the remote retrieval of a contextual fear memory. ( d ) PSD-95 GK mice froze less than WT controls during remote fear memory retrieval (n=9–10). ( e ) Schematic of experimental design for testing recent and remote retrieval of a contextual fear memory in the same mice. ( f ) PSD-95 GK mice froze less than WT controls during remote, but not recent, fear memory retrieval (n=8). Data are means ±SEM. * P
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    Image Search Results


    Postsynaptic localization of AKAP79/150 at excitatory synapses in hippocampal neurons. A, Developmental regulation of AKAP79/150 colocalization with the MAGUK PSD-95. Rat neonatal hippocampal neurons cultured at low density for the indicated number of days were stained for AKAP150 ( red ) and the postsynaptic density MAGUK PSD-95 ( green ). The small panels are magnifications of dendrites. B , Punctate colocalization of AKAP79/150 with PSD-95 on dendritic spines: proximity of AKAP79/150 puncta to presynaptic terminals stained for synapsin but not inhibitory postsynaptic elements stained for GABA A receptors. Neurons (2–3 weeks old) cultured at medium-high density were stained with anti-rat AKAP150 ( red ) and anti-PSD-95, anti-synapsin, or anti-GABA A receptor (all in green ). Codistribution is seen as yellow-orange in composite images. The red and green arrowheads point to puncta for AKAP150 and PSD-95, respectively, that are not colocalized even in “mature” dendrites. The images shown are representative of multiple neurons imaged in more than three experiments for each condition.

    Journal: The Journal of Neuroscience

    Article Title: Regulation of A-Kinase Anchoring Protein 79/150–cAMP-Dependent Protein Kinase Postsynaptic Targeting by NMDA Receptor Activation of Calcineurin and Remodeling of Dendritic Actin

    doi: 10.1523/JNEUROSCI.22-16-07027.2002

    Figure Lengend Snippet: Postsynaptic localization of AKAP79/150 at excitatory synapses in hippocampal neurons. A, Developmental regulation of AKAP79/150 colocalization with the MAGUK PSD-95. Rat neonatal hippocampal neurons cultured at low density for the indicated number of days were stained for AKAP150 ( red ) and the postsynaptic density MAGUK PSD-95 ( green ). The small panels are magnifications of dendrites. B , Punctate colocalization of AKAP79/150 with PSD-95 on dendritic spines: proximity of AKAP79/150 puncta to presynaptic terminals stained for synapsin but not inhibitory postsynaptic elements stained for GABA A receptors. Neurons (2–3 weeks old) cultured at medium-high density were stained with anti-rat AKAP150 ( red ) and anti-PSD-95, anti-synapsin, or anti-GABA A receptor (all in green ). Codistribution is seen as yellow-orange in composite images. The red and green arrowheads point to puncta for AKAP150 and PSD-95, respectively, that are not colocalized even in “mature” dendrites. The images shown are representative of multiple neurons imaged in more than three experiments for each condition.

    Article Snippet: Primary antibodies were used as follows: 1:500 rabbit anti-AKAP150, 1:1000 rabbit anti-AKAP79 (Dr. Yvonne Lai, ICOS, Bothel, WA); 1:100 mouse anti-PSD-95 (ABR, Golden, CO); 1:500 mouse anti-synapsin (Chemicon, Temulca, CA); 1:100 mouse anti-GABAA receptor (Chemicon); 1:200 anti-myc-9E10 (Santa Cruz Biotechnologies, Santa Cruz, CA); 1:100 rabbit anti-GluR1 (UBI, Lake Placid, NY); 1:200 mouse pan-anti-PSD-MAGUK family (UBI); 1:200 mouse anti-PKA-RIIβ (Transduction Labs, Los Angeles, CA); 1:500 mouse anti-CaNB (UBI).

    Techniques: Cell Culture, Staining

    Possible relationships in regulation of the AKAP79/150 scaffold and AMPA receptors by NMDA receptor signaling pathways. A – C , Implications for LTD: a model of possible roles for NMDA receptors and CaN-mediated F-actin reorganization in regulation of AKAP79/150 localization and AMPA receptor phosphorylation and localization. For the sake of simplicity, only AMPA receptor–SAP97–AKAP79/150 complexes are shown. Nearby NMDA receptor–PSD-95–AKAP79/150 complexes are also likely to participate in these signaling events. See Discussion for details.

    Journal: The Journal of Neuroscience

    Article Title: Regulation of A-Kinase Anchoring Protein 79/150–cAMP-Dependent Protein Kinase Postsynaptic Targeting by NMDA Receptor Activation of Calcineurin and Remodeling of Dendritic Actin

    doi: 10.1523/JNEUROSCI.22-16-07027.2002

    Figure Lengend Snippet: Possible relationships in regulation of the AKAP79/150 scaffold and AMPA receptors by NMDA receptor signaling pathways. A – C , Implications for LTD: a model of possible roles for NMDA receptors and CaN-mediated F-actin reorganization in regulation of AKAP79/150 localization and AMPA receptor phosphorylation and localization. For the sake of simplicity, only AMPA receptor–SAP97–AKAP79/150 complexes are shown. Nearby NMDA receptor–PSD-95–AKAP79/150 complexes are also likely to participate in these signaling events. See Discussion for details.

    Article Snippet: Primary antibodies were used as follows: 1:500 rabbit anti-AKAP150, 1:1000 rabbit anti-AKAP79 (Dr. Yvonne Lai, ICOS, Bothel, WA); 1:100 mouse anti-PSD-95 (ABR, Golden, CO); 1:500 mouse anti-synapsin (Chemicon, Temulca, CA); 1:100 mouse anti-GABAA receptor (Chemicon); 1:200 anti-myc-9E10 (Santa Cruz Biotechnologies, Santa Cruz, CA); 1:100 rabbit anti-GluR1 (UBI, Lake Placid, NY); 1:200 mouse pan-anti-PSD-MAGUK family (UBI); 1:200 mouse anti-PKA-RIIβ (Transduction Labs, Los Angeles, CA); 1:500 mouse anti-CaNB (UBI).

    Techniques:

    The AKAP79 N-terminal basic domain targets to dendritic spines in hippocampal neurons. A , Targeting of AKAP79–GFP and the N-terminal basic domain (1–153)–GFP but not GFP alone (all in green ) to dendritic spines in transfected neurons. B , Colocalization of AKAP79–GFP and (1–153)–GFP (both green ) with F-actin and PSD-95 (both red ) on dendritic spines. See Results and Materials and Methods for quantitation and details.

    Journal: The Journal of Neuroscience

    Article Title: Regulation of A-Kinase Anchoring Protein 79/150–cAMP-Dependent Protein Kinase Postsynaptic Targeting by NMDA Receptor Activation of Calcineurin and Remodeling of Dendritic Actin

    doi: 10.1523/JNEUROSCI.22-16-07027.2002

    Figure Lengend Snippet: The AKAP79 N-terminal basic domain targets to dendritic spines in hippocampal neurons. A , Targeting of AKAP79–GFP and the N-terminal basic domain (1–153)–GFP but not GFP alone (all in green ) to dendritic spines in transfected neurons. B , Colocalization of AKAP79–GFP and (1–153)–GFP (both green ) with F-actin and PSD-95 (both red ) on dendritic spines. See Results and Materials and Methods for quantitation and details.

    Article Snippet: Primary antibodies were used as follows: 1:500 rabbit anti-AKAP150, 1:1000 rabbit anti-AKAP79 (Dr. Yvonne Lai, ICOS, Bothel, WA); 1:100 mouse anti-PSD-95 (ABR, Golden, CO); 1:500 mouse anti-synapsin (Chemicon, Temulca, CA); 1:100 mouse anti-GABAA receptor (Chemicon); 1:200 anti-myc-9E10 (Santa Cruz Biotechnologies, Santa Cruz, CA); 1:100 rabbit anti-GluR1 (UBI, Lake Placid, NY); 1:200 mouse pan-anti-PSD-MAGUK family (UBI); 1:200 mouse anti-PKA-RIIβ (Transduction Labs, Los Angeles, CA); 1:500 mouse anti-CaNB (UBI).

    Techniques: Transfection, Quantitation Assay

    Analysis of the localization of synaptic proteins of neurons exposed to BAM1-EG 6 . A , representative images of dendrites (pal-GFP) PSD95 (post-synaptic marker) and Synapsin (pre-synaptic marker) from neurons dosed with control (0.1% DMSO) or BAM1-EG 6 .

    Journal: The Journal of Biological Chemistry

    Article Title: Benzothiazole Amphiphiles Promote the Formation of Dendritic Spines in Primary Hippocampal Neurons *

    doi: 10.1074/jbc.M115.701482

    Figure Lengend Snippet: Analysis of the localization of synaptic proteins of neurons exposed to BAM1-EG 6 . A , representative images of dendrites (pal-GFP) PSD95 (post-synaptic marker) and Synapsin (pre-synaptic marker) from neurons dosed with control (0.1% DMSO) or BAM1-EG 6 .

    Article Snippet: Primary antibodies used were: rabbit anti-Synapsin (EMD Millipore AB1543), mouse anti-PSD95 (EMD Millipore CP35), mouse anti-RasGRF1 (BD 610149), mouse anti-Ras (Thermo 1862335), mouse anti-GAPDH (Sigma G8795), rabbit anti-actin (Cytoskeleton AAN01), rabbit anti-Arp2 (Santa Cruz sc-15389), mouse anti-Aβ (6E10) (Biolegend® 803001), rabbit anti-p-Cofilin (Santa Cruz Biotechnology sc-12912-R) and rabbit anti-Cofilin (Santa Cruz Biotechnology sc-33779).

    Techniques: Marker

    Phosphorylation of PSD-95 by Cdk5 decreases the binding of Src to PSD-95. A , The impact of the phosphorylation of the N terminus of PSD-95 by Cdk5 on the binding of PSD-95 to Src was evaluated in transfected HEK 293T cells expressing Y527F Src (active), p35-Cdk5 (active), and either wild-type PSD-95 or the alanine mutant of PSD-95 (T19A, S25A, S35A; AAA PSD-95). Src immunoprecipitated from lysates of transfected cultures was analyzed for the amount of AAA PSD-95 coimmunoprecipitated with Src. Quantification shows that Src binds significantly more to AAA PSD-95 compared with wild-type PSD-95 (786.33 ± 156% of wild-type, p = 0.001; n = 4). B , The impact of the phosphorylation of the N terminus of PSD-95 by Cdk5 on the binding of PSD-95 to Src was evaluated in transfected HEK 293T cells expressing Y527F Src (active), p35-DNk5 (inactive), and either wild-type PSD-95 or the phosphorylation-mimic of PSD-95 (T19D, S25D, S35D; DDD PSD-95). Src immunoprecipitated from lysates of transfected cultures was analyzed for the amount of DDD PSD-95 coimmunoprecipitated with Src. Quantification shows that Src binds less to DDD PSD-95 compared with wild-type PSD-95 (38.28 ± 19.01% of wild-type; p = 0.162; n = 3).

    Journal: The Journal of Neuroscience

    Article Title: Cdk5 Regulates the Phosphorylation of Tyrosine 1472 NR2B and the Surface Expression of NMDA Receptors

    doi: 10.1523/JNEUROSCI.1900-07.2008

    Figure Lengend Snippet: Phosphorylation of PSD-95 by Cdk5 decreases the binding of Src to PSD-95. A , The impact of the phosphorylation of the N terminus of PSD-95 by Cdk5 on the binding of PSD-95 to Src was evaluated in transfected HEK 293T cells expressing Y527F Src (active), p35-Cdk5 (active), and either wild-type PSD-95 or the alanine mutant of PSD-95 (T19A, S25A, S35A; AAA PSD-95). Src immunoprecipitated from lysates of transfected cultures was analyzed for the amount of AAA PSD-95 coimmunoprecipitated with Src. Quantification shows that Src binds significantly more to AAA PSD-95 compared with wild-type PSD-95 (786.33 ± 156% of wild-type, p = 0.001; n = 4). B , The impact of the phosphorylation of the N terminus of PSD-95 by Cdk5 on the binding of PSD-95 to Src was evaluated in transfected HEK 293T cells expressing Y527F Src (active), p35-DNk5 (inactive), and either wild-type PSD-95 or the phosphorylation-mimic of PSD-95 (T19D, S25D, S35D; DDD PSD-95). Src immunoprecipitated from lysates of transfected cultures was analyzed for the amount of DDD PSD-95 coimmunoprecipitated with Src. Quantification shows that Src binds less to DDD PSD-95 compared with wild-type PSD-95 (38.28 ± 19.01% of wild-type; p = 0.162; n = 3).

    Article Snippet: This result is consistent with the increased binding of Src with PSD-95 under Cdk5 inhibition.

    Techniques: Binding Assay, Transfection, Expressing, Mutagenesis, Immunoprecipitation

    PSD-95 regulates the phosphorylation of Y1472 NR2B by Src. A , The impact of PSD-95 expression on the Src-dependent phosphorylation of Y1472 NR2B was analyzed in transfected HEK 293T cells expressing NR1/NR2B, Y527F Src (active), p35-DNk5 (inactive), with or without PSD-95. Immunoprecipitated (IP) NR2B was immunoblotted with the phospho-Y1472 NR2B-specific antibody. Quantification shows that the expression of PSD-95 increases significantly the Src-dependent phosphorylation of Y1472 NR2B by nearly fourfold (226.246 ± 41.98% compared with 60.65 ± 20.39% in lysates without PSD-95; p = 0.027; n = 5). B , The phosphorylation of Y1472 NR2B by Src was analyzed in HEK 293T cells expressing Src (Y527F), PSD-95, NR1, and either Flag-NR2B or Flag-NR2B S1480A, a mutation that dramatically decreases the binding of NR2B to the PDZ domains of PSD-95. Expression of the S1480A NR2B mutant (SA) resulted in a decrease in phosphorylation of Y1472 (41.31 ± 10.76% vs 97.99 ± 35.60% of wild-type NR2B; p = 0.142; n = 4). Wt, Wild type.

    Journal: The Journal of Neuroscience

    Article Title: Cdk5 Regulates the Phosphorylation of Tyrosine 1472 NR2B and the Surface Expression of NMDA Receptors

    doi: 10.1523/JNEUROSCI.1900-07.2008

    Figure Lengend Snippet: PSD-95 regulates the phosphorylation of Y1472 NR2B by Src. A , The impact of PSD-95 expression on the Src-dependent phosphorylation of Y1472 NR2B was analyzed in transfected HEK 293T cells expressing NR1/NR2B, Y527F Src (active), p35-DNk5 (inactive), with or without PSD-95. Immunoprecipitated (IP) NR2B was immunoblotted with the phospho-Y1472 NR2B-specific antibody. Quantification shows that the expression of PSD-95 increases significantly the Src-dependent phosphorylation of Y1472 NR2B by nearly fourfold (226.246 ± 41.98% compared with 60.65 ± 20.39% in lysates without PSD-95; p = 0.027; n = 5). B , The phosphorylation of Y1472 NR2B by Src was analyzed in HEK 293T cells expressing Src (Y527F), PSD-95, NR1, and either Flag-NR2B or Flag-NR2B S1480A, a mutation that dramatically decreases the binding of NR2B to the PDZ domains of PSD-95. Expression of the S1480A NR2B mutant (SA) resulted in a decrease in phosphorylation of Y1472 (41.31 ± 10.76% vs 97.99 ± 35.60% of wild-type NR2B; p = 0.142; n = 4). Wt, Wild type.

    Article Snippet: This result is consistent with the increased binding of Src with PSD-95 under Cdk5 inhibition.

    Techniques: Expressing, Transfection, Immunoprecipitation, Mutagenesis, Binding Assay

    Src phosphorylates NR2B on Y1472 in a Cdk5-dependent manner. A , The level of phosphorylation of NR2B at residue Y1472 was monitored in transfected HEK 293T cells expressing NR1/NR2B, PSD-95, Y527F Src (active), and either p35-cdk5 (active) or p35-DNk5 (inactive). NR2B was immunoprecipitated (IP) from the lysates of transfected cultures and probed with the phospho-Y1472 NR2B-specific antibody. Quantification shows a significant increase in Y1472 NR2B phosphorylation when dominant negative, inactive Cdk5 (p35-DNk5) was expressed compared with active Cdk5 (p35-cdk5) (257.17 ± 56.23% and 126.33 ± 30.92%, respectively; p = 0.022; n = 6). B , The level of phosphorylation of NR2B at residue Y1472 was monitored in transfected HEK 293T cells expressing a constitutively active mutant of Fyn (Y531F), PSD-95, NMDARs subunits NR1 and NR2B, and either p35-Cdk5 (active) or p35-DNk5 (inactive). Quantification of the level of phospho-Y1472 of immunoprecipitated NR2B reveals that Fyn phosphorylation of Y1472 NR2B is not increased in lysates expressing p35-DNk5 compared with those expressing p35-Cdk5 (121.65 ± 44.78% and 170.67 ± 53.42%, respectively; p = 0.194; n = 5). exp, Exposure.

    Journal: The Journal of Neuroscience

    Article Title: Cdk5 Regulates the Phosphorylation of Tyrosine 1472 NR2B and the Surface Expression of NMDA Receptors

    doi: 10.1523/JNEUROSCI.1900-07.2008

    Figure Lengend Snippet: Src phosphorylates NR2B on Y1472 in a Cdk5-dependent manner. A , The level of phosphorylation of NR2B at residue Y1472 was monitored in transfected HEK 293T cells expressing NR1/NR2B, PSD-95, Y527F Src (active), and either p35-cdk5 (active) or p35-DNk5 (inactive). NR2B was immunoprecipitated (IP) from the lysates of transfected cultures and probed with the phospho-Y1472 NR2B-specific antibody. Quantification shows a significant increase in Y1472 NR2B phosphorylation when dominant negative, inactive Cdk5 (p35-DNk5) was expressed compared with active Cdk5 (p35-cdk5) (257.17 ± 56.23% and 126.33 ± 30.92%, respectively; p = 0.022; n = 6). B , The level of phosphorylation of NR2B at residue Y1472 was monitored in transfected HEK 293T cells expressing a constitutively active mutant of Fyn (Y531F), PSD-95, NMDARs subunits NR1 and NR2B, and either p35-Cdk5 (active) or p35-DNk5 (inactive). Quantification of the level of phospho-Y1472 of immunoprecipitated NR2B reveals that Fyn phosphorylation of Y1472 NR2B is not increased in lysates expressing p35-DNk5 compared with those expressing p35-Cdk5 (121.65 ± 44.78% and 170.67 ± 53.42%, respectively; p = 0.194; n = 5). exp, Exposure.

    Article Snippet: This result is consistent with the increased binding of Src with PSD-95 under Cdk5 inhibition.

    Techniques: Transfection, Expressing, Immunoprecipitation, Dominant Negative Mutation, Mutagenesis

    Vehicle control ( left ) and short-term RXR agonist treatment ( right ) effects on total Aβ42, soluble Aβ (Aβ42 and oAβ), and PSD95 levels in E3FAD-CX, E4FAD-CX, and E3FAD-HP. EFAD mice were treated with Bex (100 mg/kg/day),

    Journal: The Journal of Biological Chemistry

    Article Title: Amyloid-β Pathology and APOE Genotype Modulate Retinoid X Receptor Agonist Activity in Vivo *

    doi: 10.1074/jbc.M114.600833

    Figure Lengend Snippet: Vehicle control ( left ) and short-term RXR agonist treatment ( right ) effects on total Aβ42, soluble Aβ (Aβ42 and oAβ), and PSD95 levels in E3FAD-CX, E4FAD-CX, and E3FAD-HP. EFAD mice were treated with Bex (100 mg/kg/day),

    Article Snippet: The following primary antibodies were used: ABCA1 (1:500, Novus Biologicals); ABCG1 (1:500, Novus Biologicals); PSD95 (1:500, Antibodies Inc.); and β-actin (1:2000, Sigma).

    Techniques: Mouse Assay

    Rivastigmine-mediated increase in neuron-associated sAPP and sAPPα parallels increases in neuronal protein markers. Levels of low molecular weight neuronal sAPP (LMW-sAPP), high molecular weight glial sAPP (HMW-sAPP), and sAPPα were compared to levels of the presynaptic protein markers SNAP-25 and syntaxin-4, and the postsynaptic protein marker PSD-95. All values are expressed as a % of vehicle-treated cells for comparison. PSD-95 and SNAP-25 levels increased dose-dependently with rivastigmine treatment (both p

    Journal: PLoS ONE

    Article Title: Rivastigmine Lowers A? and Increases sAPP? Levels, Which Parallel Elevated Synaptic Markers and Metabolic Activity in Degenerating Primary Rat Neurons

    doi: 10.1371/journal.pone.0021954

    Figure Lengend Snippet: Rivastigmine-mediated increase in neuron-associated sAPP and sAPPα parallels increases in neuronal protein markers. Levels of low molecular weight neuronal sAPP (LMW-sAPP), high molecular weight glial sAPP (HMW-sAPP), and sAPPα were compared to levels of the presynaptic protein markers SNAP-25 and syntaxin-4, and the postsynaptic protein marker PSD-95. All values are expressed as a % of vehicle-treated cells for comparison. PSD-95 and SNAP-25 levels increased dose-dependently with rivastigmine treatment (both p

    Article Snippet: Blots of the lysates were probed with mouse-anti-SNAP-25 (Millipore), mouse-anti-PSD-95 (Antibodies Incorporated, Davis, CA), mouse-anti-syntaxin-4 (BD Transduction Labs, Franklin Lakes, NJ), mouse anti-β-actin (Sigma-Aldrich), and the 22C11 antibody as above, followed by the appropriate secondary antibody and ECL detection.

    Techniques: Molecular Weight, Marker

    PSD-95 PDZ1-2 binding to SF-β1AR in supported cell membrane sheet. ( a ) Concentration dependent normalized binding of PSD-95 PDZ1-2 (black) to β 1 AR (K d * =3 ± 2), n = 3. ( b ) Representative images demonstrating a PSD95-PDZ1-2 concentration series on supported cell membranes expressing the β1-AR.

    Journal: eLife

    Article Title: Mechanisms of PDZ domain scaffold assembly illuminated by use of supported cell membrane sheets

    doi: 10.7554/eLife.39180

    Figure Lengend Snippet: PSD-95 PDZ1-2 binding to SF-β1AR in supported cell membrane sheet. ( a ) Concentration dependent normalized binding of PSD-95 PDZ1-2 (black) to β 1 AR (K d * =3 ± 2), n = 3. ( b ) Representative images demonstrating a PSD95-PDZ1-2 concentration series on supported cell membranes expressing the β1-AR.

    Article Snippet: Then labelled with mouse anti-PSD95 (Antibodies inc.#72–028) 1:500 and rabbit anti-GFP (abcam #ab290) 1:500 for 1 hr at room temperature.

    Techniques: Binding Assay, Concentration Assay, Expressing

    CNK2 interacts with TNIK and the two proteins co-localise in dendritic spines. ( A ) Scheme of CNK1 and CNK2 domain architecture (SAM, sterile alpha motif; CRIC, conserved region in CNK; PDZ, PSD-95/DLG-1/ZO-1; DUF1170, domain of unknown function; PH, pleckstrin homology). ( B ) CNK2 specifically interacts with TNIK. Coimmunoprecipitation experiment with CNK1-V5, CNK2-V5 and FLAG-TNIK expressed in CHL V79 cells. Proteins were immunoprecipitated with either anti-V5 (mouse) antibody or mouse IgGs as a negative control. Proteins were detected by western blot with anti-V5 (CNK2) and anti-FLAG (TNIK) antibodies. ( C ) Representative image of a primary neuron (DIV 21) showing endogenous CNK2 (cyan) and endogenous TNIK (magenta) in dendritic spines. Scale bar: 10 µm ( D ) Coimmunoprecipitation experiment of EGFP-CNK2 with endogenous TNIK expressed in neurons. Protein was immunoprecipitated with either anti-GFP (mouse) or mouse IgGs as a negative control. Proteins were detected by western blot with anti-GFP (CNK2) and anti-TNIK antibodies. Input control (lysate) is shown on the left.

    Journal: Scientific Reports

    Article Title: Disease-associated synaptic scaffold protein CNK2 modulates PSD size and influences localisation of the regulatory kinase TNIK

    doi: 10.1038/s41598-020-62207-4

    Figure Lengend Snippet: CNK2 interacts with TNIK and the two proteins co-localise in dendritic spines. ( A ) Scheme of CNK1 and CNK2 domain architecture (SAM, sterile alpha motif; CRIC, conserved region in CNK; PDZ, PSD-95/DLG-1/ZO-1; DUF1170, domain of unknown function; PH, pleckstrin homology). ( B ) CNK2 specifically interacts with TNIK. Coimmunoprecipitation experiment with CNK1-V5, CNK2-V5 and FLAG-TNIK expressed in CHL V79 cells. Proteins were immunoprecipitated with either anti-V5 (mouse) antibody or mouse IgGs as a negative control. Proteins were detected by western blot with anti-V5 (CNK2) and anti-FLAG (TNIK) antibodies. ( C ) Representative image of a primary neuron (DIV 21) showing endogenous CNK2 (cyan) and endogenous TNIK (magenta) in dendritic spines. Scale bar: 10 µm ( D ) Coimmunoprecipitation experiment of EGFP-CNK2 with endogenous TNIK expressed in neurons. Protein was immunoprecipitated with either anti-GFP (mouse) or mouse IgGs as a negative control. Proteins were detected by western blot with anti-GFP (CNK2) and anti-TNIK antibodies. Input control (lysate) is shown on the left.

    Article Snippet: Antibodies Primary antibodies: anti-CNK2 (guinea pig, Eurogentec, custom-made), anti-CNK2 (rabbit, Sigma/Atlas, HPA 001502), anti-Homer-1 (guinea pig, Synaptic Systems, 160004), anti-MAP2 (guinea pig, Synaptic Systems, 188004), anti-MAP2 (mouse, Millipore, 05–346), anti-Mortalin (mouse, Antibodies Inc.,75–127), anti-PSD-95 (mouse, Antibodies Inc., 75–028), anti-SYNAPSIN-1 (rabbit, Synaptic systems, 106 103), anti-TNIK (mouse, Santa Cruz, sc-136103), anti-FLAG-HRP (mouse, Sigma), anti-GFP (chicken, Abcam, ab13970), anti-GFP (mouse, Roche, 11814460001), anti-GFP (goat, Abcam, ab6673), normal mouse IgG (sc-2025, Santa Cruz), anti-V5 (mouse, Invitrogen, R960–25), anti-V5 (rabbit, Millipore, AB3792).

    Techniques: Immunoprecipitation, Negative Control, Western Blot

    CNK2 is a membrane-associated protein and localised in the post-synapse of neurons. ( A ) CNK2 is expressed from P0 throughout adulthood in the mouse brain. Whole cell lysates generated from mouse brains at P0, P10, P20 and adult stage were analysed by SDS-PAGE and western blot with anti-CNK2 antibody. Mortalin serves as loading control (expected size 60 kDa). ( B ) Membrane localisation of overexpressed EGFP-tagged CNK2 (EGFP-CNK2) in CHL V79 cells (EGFP-CNK2 cyan; DAPI blue). Scale bar: 20 µm. ( C ) Localisation of endogenous CNK2 (cyan) and the dendritic marker MAP2 (magenta) in cultured rat hippocampal neurons (DIV21). Endogenous CNK2 is localised in punctate structures along the dendrite. Scale bar: 20 µm ( D ) Localisation of EGFP-CNK2 (cyan) following lentiviral transduction, and the dendritic marker MAP2 (magenta) in cultured rat hippocampal neurons (DIV21). Scale bar: 20 µm. ( E ) CNK2 (cyan) colocalises with postsynaptic marker PSD-95 (magenta). ( F ) CNK2 (cyan) shows adjacent staining with the presynaptic marker Synapsin (magenta). Scale bar: 10 µm.

    Journal: Scientific Reports

    Article Title: Disease-associated synaptic scaffold protein CNK2 modulates PSD size and influences localisation of the regulatory kinase TNIK

    doi: 10.1038/s41598-020-62207-4

    Figure Lengend Snippet: CNK2 is a membrane-associated protein and localised in the post-synapse of neurons. ( A ) CNK2 is expressed from P0 throughout adulthood in the mouse brain. Whole cell lysates generated from mouse brains at P0, P10, P20 and adult stage were analysed by SDS-PAGE and western blot with anti-CNK2 antibody. Mortalin serves as loading control (expected size 60 kDa). ( B ) Membrane localisation of overexpressed EGFP-tagged CNK2 (EGFP-CNK2) in CHL V79 cells (EGFP-CNK2 cyan; DAPI blue). Scale bar: 20 µm. ( C ) Localisation of endogenous CNK2 (cyan) and the dendritic marker MAP2 (magenta) in cultured rat hippocampal neurons (DIV21). Endogenous CNK2 is localised in punctate structures along the dendrite. Scale bar: 20 µm ( D ) Localisation of EGFP-CNK2 (cyan) following lentiviral transduction, and the dendritic marker MAP2 (magenta) in cultured rat hippocampal neurons (DIV21). Scale bar: 20 µm. ( E ) CNK2 (cyan) colocalises with postsynaptic marker PSD-95 (magenta). ( F ) CNK2 (cyan) shows adjacent staining with the presynaptic marker Synapsin (magenta). Scale bar: 10 µm.

    Article Snippet: Antibodies Primary antibodies: anti-CNK2 (guinea pig, Eurogentec, custom-made), anti-CNK2 (rabbit, Sigma/Atlas, HPA 001502), anti-Homer-1 (guinea pig, Synaptic Systems, 160004), anti-MAP2 (guinea pig, Synaptic Systems, 188004), anti-MAP2 (mouse, Millipore, 05–346), anti-Mortalin (mouse, Antibodies Inc.,75–127), anti-PSD-95 (mouse, Antibodies Inc., 75–028), anti-SYNAPSIN-1 (rabbit, Synaptic systems, 106 103), anti-TNIK (mouse, Santa Cruz, sc-136103), anti-FLAG-HRP (mouse, Sigma), anti-GFP (chicken, Abcam, ab13970), anti-GFP (mouse, Roche, 11814460001), anti-GFP (goat, Abcam, ab6673), normal mouse IgG (sc-2025, Santa Cruz), anti-V5 (mouse, Invitrogen, R960–25), anti-V5 (rabbit, Millipore, AB3792).

    Techniques: Generated, SDS Page, Western Blot, Marker, Cell Culture, Transduction, Staining

    ( A ) Left: Representative immunoblot of membrane-enriched, Triton-X insoluble whole-brain homogenate lysates from young (1.5 mo) and aged ( > 2 years) WT FVB mice, separated on a 4–18% Tris-HCl acrylamide gel, and probed for PSD-95. Right: densitometric quantifications of PSD-95 normalized by GAPDH, expressed as folds of 1.5 mo (***p

    Journal: Scientific Reports

    Article Title: Lipid-dependent deposition of alpha-synuclein and Tau on neuronal Secretogranin II-positive vesicular membranes with age

    doi: 10.1038/s41598-018-33474-z

    Figure Lengend Snippet: ( A ) Left: Representative immunoblot of membrane-enriched, Triton-X insoluble whole-brain homogenate lysates from young (1.5 mo) and aged ( > 2 years) WT FVB mice, separated on a 4–18% Tris-HCl acrylamide gel, and probed for PSD-95. Right: densitometric quantifications of PSD-95 normalized by GAPDH, expressed as folds of 1.5 mo (***p

    Article Snippet: Primary antibodies included antibodies to aSYN (syn-1, 1:1,000, BD Biosciences, 610787 (RRID:AB_398107); H3C, 2 µg/mL, Development Studies Hybridoma Bank) (RRID:AB_2618046), total Tau (1:5,000, DAKO, A0024) (RRID:AB_10013724), phospho-Tau (AT-8, 1:2,000, Thermo Fisher Scientific, MN1020) (RRID:AB_1288949), GAPDH (1:2,000; Milipore Sigma, AB2302) (RRID:AB_10615768), TUBB3 (1:2,000, Covance, MRB435P) (RRID:AB_291636), PSD-95 (1:1,000, BD Biosciences, 610495) (RRID:AB_397861), Synapsin-1 (1:1,000, Chemicon, AB1543P) (RRID:AB_212517).

    Techniques: Mouse Assay, Acrylamide Gel Assay

    (A) Representative Western blots and densitometry analysis of GLT1, PSD-95, and SNAP-25 following 7 days treatment with Aβo, oleocanthal, or combination. Aβo significantly reduced GLT1 and PSD-95 expressions that were rectified by oleocanthal to levels above their baseline expressions; SNAP-25 was not significantly altered by the 7 days exposure to Aβo. (B) Representative Western blots and densitometry analysis of APP, sAPPα, and sAPPβ; 7 days exposure to Aβo significantly increased APP, sAPPα, and sAPPβ levels that were not modulated by oleocanthal. LRP1 was not significantly altered by the treatments. Data is presented as mean ± SD (*P

    Journal: Neuroscience

    Article Title: Oleocanthal Ameliorates Amyloid-β Oligomers Toxicity on Astrocytes and Neuronal Cells: In-vitro Studies

    doi: 10.1016/j.neuroscience.2017.03.059

    Figure Lengend Snippet: (A) Representative Western blots and densitometry analysis of GLT1, PSD-95, and SNAP-25 following 7 days treatment with Aβo, oleocanthal, or combination. Aβo significantly reduced GLT1 and PSD-95 expressions that were rectified by oleocanthal to levels above their baseline expressions; SNAP-25 was not significantly altered by the 7 days exposure to Aβo. (B) Representative Western blots and densitometry analysis of APP, sAPPα, and sAPPβ; 7 days exposure to Aβo significantly increased APP, sAPPα, and sAPPβ levels that were not modulated by oleocanthal. LRP1 was not significantly altered by the treatments. Data is presented as mean ± SD (*P

    Article Snippet: We measured expression of the synaptic proteins GLT1, PSD-95, and SNAP-25.

    Techniques: Western Blot

    The relative expression of neuronal synaptic proteins GLT1, PSD-95, and SNAP-25 were compared in SH-SY5Y-APP and non-transfected SH-SY5Y cells using Western blot. Densitometry analysis of GLT1, PSD-95, and SNAP-25 revealed significantly lower expressions in SH-SY5Y-APP cells compared to SH-SY5Y cells. Data is presented as mean ± SD (***P

    Journal: Neuroscience

    Article Title: Oleocanthal Ameliorates Amyloid-β Oligomers Toxicity on Astrocytes and Neuronal Cells: In-vitro Studies

    doi: 10.1016/j.neuroscience.2017.03.059

    Figure Lengend Snippet: The relative expression of neuronal synaptic proteins GLT1, PSD-95, and SNAP-25 were compared in SH-SY5Y-APP and non-transfected SH-SY5Y cells using Western blot. Densitometry analysis of GLT1, PSD-95, and SNAP-25 revealed significantly lower expressions in SH-SY5Y-APP cells compared to SH-SY5Y cells. Data is presented as mean ± SD (***P

    Article Snippet: We measured expression of the synaptic proteins GLT1, PSD-95, and SNAP-25.

    Techniques: Expressing, Transfection, Western Blot

    Representative Western blots and densitometry analysis of GLT1, PSD-95, and SNAP-25 in SH-SY5Y after 3 and 7 days treatment with Aβo, oleocanthal, or combination. None of the treatments altered these proteins expression in SH-SY5Y cells. Data is presented as mean ± SD, n= 3 independent experiments.

    Journal: Neuroscience

    Article Title: Oleocanthal Ameliorates Amyloid-β Oligomers Toxicity on Astrocytes and Neuronal Cells: In-vitro Studies

    doi: 10.1016/j.neuroscience.2017.03.059

    Figure Lengend Snippet: Representative Western blots and densitometry analysis of GLT1, PSD-95, and SNAP-25 in SH-SY5Y after 3 and 7 days treatment with Aβo, oleocanthal, or combination. None of the treatments altered these proteins expression in SH-SY5Y cells. Data is presented as mean ± SD, n= 3 independent experiments.

    Article Snippet: We measured expression of the synaptic proteins GLT1, PSD-95, and SNAP-25.

    Techniques: Western Blot, Expressing

    1NM-PP1 suppresses punctate distribution of PSD-95 dose-dependently in TrkB F616A neurons. (A) The top row shows representative neurons immunostainted with anti-PSD-95. Typical secondary dendritic branches analyzed are shown in 48 μm 2 rectangles in the middle row. These branches are processed after the thresholding shown in the bottom row. The scales in the top and bottom rows show 10 and 1 μm, respectively. (B) Graph showing averaged PSD-95 intensities in somata. (C) Graph showing quantification of PSD-95 puncta total pixel intensities. Note that all three concentrations of 1NM-PP1 result in reduced total PSD-95 puncta intensity. (D) In this graph, PSD-95 puncta intensities are normalized to somal PSD-95 intensities. In each condition, 16 branches from eight cells (chosen from the two different dissociations) were analyzed. Error bars represent SEM.

    Journal: Frontiers in Synaptic Neuroscience

    Article Title: Postsynaptic localization of PSD-95 is regulated by all three pathways downstream of TrkB signaling

    doi: 10.3389/fnsyn.2014.00006

    Figure Lengend Snippet: 1NM-PP1 suppresses punctate distribution of PSD-95 dose-dependently in TrkB F616A neurons. (A) The top row shows representative neurons immunostainted with anti-PSD-95. Typical secondary dendritic branches analyzed are shown in 48 μm 2 rectangles in the middle row. These branches are processed after the thresholding shown in the bottom row. The scales in the top and bottom rows show 10 and 1 μm, respectively. (B) Graph showing averaged PSD-95 intensities in somata. (C) Graph showing quantification of PSD-95 puncta total pixel intensities. Note that all three concentrations of 1NM-PP1 result in reduced total PSD-95 puncta intensity. (D) In this graph, PSD-95 puncta intensities are normalized to somal PSD-95 intensities. In each condition, 16 branches from eight cells (chosen from the two different dissociations) were analyzed. Error bars represent SEM.

    Article Snippet: Therefore, we predict that MAPK/ERK regulates either or both transcription and translation of PSD-95.

    Techniques:

    PSD-95 puncta intensities in neurons that are treated with inhibitors of signaling molecules downstream of TrkB. Neurons are treated with blockers of the PLC (U73122, 1 μM), MAPK (PD98059, 50 μM), or PI3K (Wortmannin, 100 nM). Quantification was performed in the same manner as data presented in Figure 2 . The graphs show averaged PSD-95 intensities in somata (A) , total pixel intensities of PSD-95 puncta (B) , and puncta intensities normalized to somal intensities (C) . In each condition, 16 branches from eight cells were analyzed. Error bars represent SEM.

    Journal: Frontiers in Synaptic Neuroscience

    Article Title: Postsynaptic localization of PSD-95 is regulated by all three pathways downstream of TrkB signaling

    doi: 10.3389/fnsyn.2014.00006

    Figure Lengend Snippet: PSD-95 puncta intensities in neurons that are treated with inhibitors of signaling molecules downstream of TrkB. Neurons are treated with blockers of the PLC (U73122, 1 μM), MAPK (PD98059, 50 μM), or PI3K (Wortmannin, 100 nM). Quantification was performed in the same manner as data presented in Figure 2 . The graphs show averaged PSD-95 intensities in somata (A) , total pixel intensities of PSD-95 puncta (B) , and puncta intensities normalized to somal intensities (C) . In each condition, 16 branches from eight cells were analyzed. Error bars represent SEM.

    Article Snippet: Therefore, we predict that MAPK/ERK regulates either or both transcription and translation of PSD-95.

    Techniques: Planar Chromatography

    Zeta inhibitory peptide (ZIP) but not PKMζ knockdown causes suppression in PSD-95 puncta intensity. (A) Neurons are treated with ZIP (1 μM), PKC (Chelerythrine, 2.5 μM), or the scrambled ZIP peptide (1 μM). (B) Neurons are transfected with DNA constructs encoding siRNA for PKMϖ or the scrambled sequence. In both experiments, 16 branches from eight cells were analyzed. Error bars represent SEM.

    Journal: Frontiers in Synaptic Neuroscience

    Article Title: Postsynaptic localization of PSD-95 is regulated by all three pathways downstream of TrkB signaling

    doi: 10.3389/fnsyn.2014.00006

    Figure Lengend Snippet: Zeta inhibitory peptide (ZIP) but not PKMζ knockdown causes suppression in PSD-95 puncta intensity. (A) Neurons are treated with ZIP (1 μM), PKC (Chelerythrine, 2.5 μM), or the scrambled ZIP peptide (1 μM). (B) Neurons are transfected with DNA constructs encoding siRNA for PKMϖ or the scrambled sequence. In both experiments, 16 branches from eight cells were analyzed. Error bars represent SEM.

    Article Snippet: Therefore, we predict that MAPK/ERK regulates either or both transcription and translation of PSD-95.

    Techniques: Transfection, Construct, Sequencing

    Morphine decreases the phosphorylation of GluR1 at Ser 845 and inhibits the interaction between GluR1 and PSD-95 through μ-opioid receptor. (A) Neurons were treated with no drug (Ctl) or 10 µM morphine for various durations (3 hours, 6 hours and 1 day) (from left to right). The cell lysates were immunoprecipitated with an anti-GluR1 antibody. The amount of phosphorylated GluR1 at Ser 845 (Phospho-S845; first ), PSD95 proteins ( second ) and total GluR1 subunits ( third ) in the immunoprecipitation complex were detected with appropriate antibodies. Total GluR1 from whole cell extracts (Input) was also determined ( fourth ). (B) The western blots in (A) were quantified with densitometry and normalized to the untreated control ( n = 5). Note that the amount of PSD95 immunoprecipitated by the anti-GluR1 antibody significantly decreases at 6 hours and 1 day after addition of morphine, indicating a dissociation between the postsynaptic density and GluR1 subunits upon drug treatment. (C) Neurons were treated with no drug (Ctl) or 10 µM morphine for 6 hours and 1 day in the absence (vehicle) or presence of 10 µM CTOP. The cell lysates were immunoprecipitated with an anti-GluR1 antibody. The amount of GluR1-S845 phosphorylation (Phospho-S845; first ), PSD95 ( second ) and total GluR1 ( third ) in the immunoprecipitation complex was detected. Total GluR1 subunits from whole cell lysates (Input) were also determined ( fourth ). (D) and ( E ) show densitometric quantifications of western blots in (C) on the amount of phospho-S845 GluR1 and PSD-95 in the receptor complex, respectively (normalized to the untreated control; n = 3). * p

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Morphine induces AMPA receptor internalization in primary hippocampal neurons via calcineurin-dependent dephosphorylation of GluR1 subunits

    doi: 10.1523/JNEUROSCI.4255-10.2010

    Figure Lengend Snippet: Morphine decreases the phosphorylation of GluR1 at Ser 845 and inhibits the interaction between GluR1 and PSD-95 through μ-opioid receptor. (A) Neurons were treated with no drug (Ctl) or 10 µM morphine for various durations (3 hours, 6 hours and 1 day) (from left to right). The cell lysates were immunoprecipitated with an anti-GluR1 antibody. The amount of phosphorylated GluR1 at Ser 845 (Phospho-S845; first ), PSD95 proteins ( second ) and total GluR1 subunits ( third ) in the immunoprecipitation complex were detected with appropriate antibodies. Total GluR1 from whole cell extracts (Input) was also determined ( fourth ). (B) The western blots in (A) were quantified with densitometry and normalized to the untreated control ( n = 5). Note that the amount of PSD95 immunoprecipitated by the anti-GluR1 antibody significantly decreases at 6 hours and 1 day after addition of morphine, indicating a dissociation between the postsynaptic density and GluR1 subunits upon drug treatment. (C) Neurons were treated with no drug (Ctl) or 10 µM morphine for 6 hours and 1 day in the absence (vehicle) or presence of 10 µM CTOP. The cell lysates were immunoprecipitated with an anti-GluR1 antibody. The amount of GluR1-S845 phosphorylation (Phospho-S845; first ), PSD95 ( second ) and total GluR1 ( third ) in the immunoprecipitation complex was detected. Total GluR1 subunits from whole cell lysates (Input) were also determined ( fourth ). (D) and ( E ) show densitometric quantifications of western blots in (C) on the amount of phospho-S845 GluR1 and PSD-95 in the receptor complex, respectively (normalized to the untreated control; n = 3). * p

    Article Snippet: Altogether, the morphine-decreased phosphorylation of GluR1-S845 and -induced dissociation from PSD-95 indicate a probable mechanism for the opioid agonist to regulate the AMPAR trafficking.

    Techniques: CTL Assay, Immunoprecipitation, Western Blot

    Protein levels of glutamatergic markers in the lateral amygdala of human heroin and control subjects from the heroin abuse population (Study II). A. Representative WB images of GluA1 (106 kD), PSD-95 (95 kD), mGluR5 (130 kD), Homer 1b/c (45 kD), GluN1 (110) kD and GAPDH (38 kD) in two control and two heroin subjects. B. Comparison of the immunoreactivities between human heroin (n=27–28) and control subjects (n=8–13). Protein levels are presented as percent of mean control values (mean ± SEM). *, p

    Journal: Biological psychiatry

    Article Title: Dysregulated post-synaptic density and endocytic zone in the amygdala of human heroin and cocaine abusers

    doi: 10.1016/j.biopsych.2010.09.037

    Figure Lengend Snippet: Protein levels of glutamatergic markers in the lateral amygdala of human heroin and control subjects from the heroin abuse population (Study II). A. Representative WB images of GluA1 (106 kD), PSD-95 (95 kD), mGluR5 (130 kD), Homer 1b/c (45 kD), GluN1 (110) kD and GAPDH (38 kD) in two control and two heroin subjects. B. Comparison of the immunoreactivities between human heroin (n=27–28) and control subjects (n=8–13). Protein levels are presented as percent of mean control values (mean ± SEM). *, p

    Article Snippet: Identification of PSD-95 as a regulator of dopamine-mediated synaptic and behavioral plasticity.

    Techniques: Western Blot

    Immunoblots of NR1, NR2A, and actin (a) nNOS, PSD-95, and actin (b) as well as glutamine synthetase (GS), GAD67, and tubulin (c) from 3 representative pairs of controls (Cont) and depressed subjects (Dep) used in the analysis. Each well was loaded with

    Journal:

    Article Title: Elevated levels of NR2A and PSD-95 in the lateral amygdala in depression

    doi: 10.1017/S1461145708008985

    Figure Lengend Snippet: Immunoblots of NR1, NR2A, and actin (a) nNOS, PSD-95, and actin (b) as well as glutamine synthetase (GS), GAD67, and tubulin (c) from 3 representative pairs of controls (Cont) and depressed subjects (Dep) used in the analysis. Each well was loaded with

    Article Snippet: PSD-95 and nNOS immunoreactivities appeared as bands with molecular masses of 95 and 160 kDa, respectively ( ).

    Techniques: Western Blot

    Amounts of NR1, NR2A, PSD-95, and nNOS immunoreactivities in the lateral amygdala from control subjects (open circles; n=13-14) and depressed subjects (filled circles; n=13-14). Significant increases in the NR2A and PSD-95 immunoreactivities (+115% and

    Journal:

    Article Title: Elevated levels of NR2A and PSD-95 in the lateral amygdala in depression

    doi: 10.1017/S1461145708008985

    Figure Lengend Snippet: Amounts of NR1, NR2A, PSD-95, and nNOS immunoreactivities in the lateral amygdala from control subjects (open circles; n=13-14) and depressed subjects (filled circles; n=13-14). Significant increases in the NR2A and PSD-95 immunoreactivities (+115% and

    Article Snippet: PSD-95 and nNOS immunoreactivities appeared as bands with molecular masses of 95 and 160 kDa, respectively ( ).

    Techniques:

    Subsynaptic localization of dysbindin-1 isoforms. A : Western blotting results on synaptosomal (Syn), synaptic vesicle (SV), presynaptic membrane (PrS), and postsynaptic density (PSD) fractions of the normal HF from three control cases (1–3) in this study. Successful synaptic fractionation was confirmed with synaptophysin as a marker for synaptic vesicles and PSD-95 as a marker for the PSD. No selective molecular marker is available for the PrS fraction, including syntaxin-1 since this proves to be ubiquitously distributed on subcellular membranes. β-actin served as the loading control. The blots were probed with PA3111 (1∶1000) for dysbindin-1A and -1C and with UPenn 331 (1∶5000) for dysbindin-1B. In synaptosomes, dysbindin-1A and -1C were found to be predominantly PSD proteins, while dysbindin-1B was found to be predominantly a synaptic vesicle-associated protein. MW = molecular weight marker position. B : Diagram of pre- and/or post-synaptic location of dysbindin-1 isoforms consistent with the Western blotting results just shown and with dysbindin-1 immunohistochemical findings at the electron microscopic level by Talbot et al. [43] .

    Journal: PLoS ONE

    Article Title: Synaptic Dysbindin-1 Reductions in Schizophrenia Occur in an Isoform-Specific Manner Indicating Their Subsynaptic Location

    doi: 10.1371/journal.pone.0016886

    Figure Lengend Snippet: Subsynaptic localization of dysbindin-1 isoforms. A : Western blotting results on synaptosomal (Syn), synaptic vesicle (SV), presynaptic membrane (PrS), and postsynaptic density (PSD) fractions of the normal HF from three control cases (1–3) in this study. Successful synaptic fractionation was confirmed with synaptophysin as a marker for synaptic vesicles and PSD-95 as a marker for the PSD. No selective molecular marker is available for the PrS fraction, including syntaxin-1 since this proves to be ubiquitously distributed on subcellular membranes. β-actin served as the loading control. The blots were probed with PA3111 (1∶1000) for dysbindin-1A and -1C and with UPenn 331 (1∶5000) for dysbindin-1B. In synaptosomes, dysbindin-1A and -1C were found to be predominantly PSD proteins, while dysbindin-1B was found to be predominantly a synaptic vesicle-associated protein. MW = molecular weight marker position. B : Diagram of pre- and/or post-synaptic location of dysbindin-1 isoforms consistent with the Western blotting results just shown and with dysbindin-1 immunohistochemical findings at the electron microscopic level by Talbot et al. [43] .

    Article Snippet: Primary antibodies were used at dilutions that allowed detection of their targets in a linear quantitative range: β-actin (Sigma Aldrich A3853 at 1∶4000), dysbindin-1A and 1C (Hdys764 at 1∶800; PA3111 at 1∶800–1∶1000, or UPenn 329 at 1∶200), dysbindin-1B (UPenn 331 at 1∶1000–1∶5000), PSD-95 (US Biological, Swampscott, MA, USA, P9150-10 at 1∶1000), and synaptophysin (Millipore, Billerica, MA, USA, MSB5258 at 1∶5000).

    Techniques: Western Blot, Fractionation, Marker, Molecular Weight, Immunohistochemistry

    Knock-down of Tm-agrin in mature hippocampal neurons reduces synapse density Hippocampal neurons fixed at DIV21, 7 days post infection, were immuno-stained for PSD-95 (green) to visualize post-synaptic puncta and synaptogamin (red) to label presynaptic boutons. All dendrites were measured and all synapses were counted as described above. Examples of hippocampal neurons from (A) uninfected control, (B) VsiMock-infected and (C) VsiAgrin-infected cultures are shown. Yellow arrowheads indicate examples of overlapping puncta of PSD-95 and synaptotagmin that were counted as synapses. (D) Quantitative assay of synapse density in each group demonstrates that the VsiAgrin-infected neurons had a significantly lower number of synapses per 100μm dendritic length compared to control or VsiMock-infected neurons. Synapse density of VsiMock-infected neurons was not significantly different from uninfected control (N=75 neurons, 3 separate experiments). Bars show the mean ± SEM. Asterisks indicate value significantly different from VsiMock and uninfected control. Bar = 50 μm.

    Journal: Neuroscience

    Article Title: Transmembrane Agrin Regulates Dendritic Filopodia and Synapse Formation in Mature Hippocampal Neuron Cultures

    doi: 10.1016/j.neuroscience.2009.06.012

    Figure Lengend Snippet: Knock-down of Tm-agrin in mature hippocampal neurons reduces synapse density Hippocampal neurons fixed at DIV21, 7 days post infection, were immuno-stained for PSD-95 (green) to visualize post-synaptic puncta and synaptogamin (red) to label presynaptic boutons. All dendrites were measured and all synapses were counted as described above. Examples of hippocampal neurons from (A) uninfected control, (B) VsiMock-infected and (C) VsiAgrin-infected cultures are shown. Yellow arrowheads indicate examples of overlapping puncta of PSD-95 and synaptotagmin that were counted as synapses. (D) Quantitative assay of synapse density in each group demonstrates that the VsiAgrin-infected neurons had a significantly lower number of synapses per 100μm dendritic length compared to control or VsiMock-infected neurons. Synapse density of VsiMock-infected neurons was not significantly different from uninfected control (N=75 neurons, 3 separate experiments). Bars show the mean ± SEM. Asterisks indicate value significantly different from VsiMock and uninfected control. Bar = 50 μm.

    Article Snippet: Primary antibodies used were mouse monoclonal antibody to PSD-95 (1:2000) (DakoCytomation, Inc., Carpinteria, CA), mouse monoclonal antibody to MAP2 (1:2000) (Chemicon International, Temecula, CA), rabbit antiserum to γ-actin (1:1000) (kindly provided by Dr. J. Bulinsky); rabbit antiserum to synaptotagmin I (V216; 1:1000) (kindly provided by Dr. T. Sudhof) and chicken monoclonal antibody to GFP (1:1500) (Aves Labs, Inc., Tigard, Or).

    Techniques: Infection, Staining

    Depletion of Tm-agrin in dendrites or axons alone reduces synapse density Hippocampal neuron cultures were infected with a reduced concentration of shRNA lentivirus to obtain an infection rate of approximately 40%. This allowed the identification of regions of contact between axons and dendrites where either the axon or the dendrite, but not both, emanated from an infected neuron. In each 4-part panel, X’ shows GFP immuno-staining (blue in merge, indicting virus infected axons or dendrites), X” shows PSD-95 (green in merge) and X”’ shows synaptotagmin (SYN) red in merge) in the same field. Yellow arrowheads indicate examples of counted synapses. (A) Example of axon(s) from a VsiMock-infected neuron penetrating an uninfected dendritic network. Synapse formation is unaffected, with numerous PSD-95 and synaptotagmin puncta overlapping along the regions of contact between infected axons and uninfected dendrites. (B) In contrast, a VsiAgrin-infected axon interacting with an uninfected dendritic network shows relatively few overlapping puncta, although many PSD-95 (green in merge) puncta are present. In the same field many synapses are seen where dendrites from the same uninfected neuron are contacted by axons of an uninfected neuron. (C) Part of the dendritic arbor of a VsiMock-infected neuron innervated by axons from uninfected neurons, displaying a typical density of synapses. (D) In contrast, the dendritic arbor of a VsiAgrin-infected neuron displays a low density of synapses. Bar = 10 μm.

    Journal: Neuroscience

    Article Title: Transmembrane Agrin Regulates Dendritic Filopodia and Synapse Formation in Mature Hippocampal Neuron Cultures

    doi: 10.1016/j.neuroscience.2009.06.012

    Figure Lengend Snippet: Depletion of Tm-agrin in dendrites or axons alone reduces synapse density Hippocampal neuron cultures were infected with a reduced concentration of shRNA lentivirus to obtain an infection rate of approximately 40%. This allowed the identification of regions of contact between axons and dendrites where either the axon or the dendrite, but not both, emanated from an infected neuron. In each 4-part panel, X’ shows GFP immuno-staining (blue in merge, indicting virus infected axons or dendrites), X” shows PSD-95 (green in merge) and X”’ shows synaptotagmin (SYN) red in merge) in the same field. Yellow arrowheads indicate examples of counted synapses. (A) Example of axon(s) from a VsiMock-infected neuron penetrating an uninfected dendritic network. Synapse formation is unaffected, with numerous PSD-95 and synaptotagmin puncta overlapping along the regions of contact between infected axons and uninfected dendrites. (B) In contrast, a VsiAgrin-infected axon interacting with an uninfected dendritic network shows relatively few overlapping puncta, although many PSD-95 (green in merge) puncta are present. In the same field many synapses are seen where dendrites from the same uninfected neuron are contacted by axons of an uninfected neuron. (C) Part of the dendritic arbor of a VsiMock-infected neuron innervated by axons from uninfected neurons, displaying a typical density of synapses. (D) In contrast, the dendritic arbor of a VsiAgrin-infected neuron displays a low density of synapses. Bar = 10 μm.

    Article Snippet: Primary antibodies used were mouse monoclonal antibody to PSD-95 (1:2000) (DakoCytomation, Inc., Carpinteria, CA), mouse monoclonal antibody to MAP2 (1:2000) (Chemicon International, Temecula, CA), rabbit antiserum to γ-actin (1:1000) (kindly provided by Dr. J. Bulinsky); rabbit antiserum to synaptotagmin I (V216; 1:1000) (kindly provided by Dr. T. Sudhof) and chicken monoclonal antibody to GFP (1:1500) (Aves Labs, Inc., Tigard, Or).

    Techniques: Infection, Concentration Assay, shRNA, Immunostaining

    Changes in the relative number of filopodia, spines, dendrite branches and synapses during development in culture (A) Fixed hippocampal neurons were immuno-stained for γ-actin (green), to visualize filopodia and spines and MAP2 (red) to highlight the dendritic arbor. Examples shown here are at 7 days in vitro (DIV) and 21 DIV. Arrows indicate examples of filopodia; arrowheads indicate examples of spines. The inset in the right-hand panel is an ∼ 3-fold enlargement of the boxed area. (B) Fixed hippocampal neurons were immuno-stained for PSD-95 (green), to visualize post-synaptic sites and synaptogamin (red) to highlight the pre-synaptic boutons. Overlapping puncta of PSD-95 and synaptotagmin (yellow) were considered to be synapses. Examples shown here are at 7 DIV and 21 DIV. Yellow arrowheads indicate examples of synapses. Arrows indicate examples or PSD-95 puncta without overlapping synaptotagmin puncta. Bar = 10 μm. (C) Graph illustrating the number of filopodia /100μm dendrite length comparing DIV 7, 14 and 21 hippocampal neurons (n=100 dendrites at least 100 μm long, from at least 25 separate neurons at DIV7, 14 or 21; 3 separate experiments). As the neurons mature, the density of filopodia declines. (D-F) Graphs illustrating the number of spines /100μm dendrite length (D), dendritic branches per neuron (E) and the number of synapses /100μm dendrite length (F), comparing DIV 7, 14 and 21 hippocampal neurons. In C, D and F, n=100 dendrites at least 100 μm long from at least 25 separate neurons at DIV 7, 14 or 21; 3 separate experiments. In E, n= at least 25 neurons at DIV 7, 14 or 21; 3 separate experiments). As the neurons mature, the density of synapses increases 15 fold, similar to the increase observed with spines, while branching of the dendritic arbor increases markedly. All data bars represent the mean ± SEM.

    Journal: Neuroscience

    Article Title: Transmembrane Agrin Regulates Dendritic Filopodia and Synapse Formation in Mature Hippocampal Neuron Cultures

    doi: 10.1016/j.neuroscience.2009.06.012

    Figure Lengend Snippet: Changes in the relative number of filopodia, spines, dendrite branches and synapses during development in culture (A) Fixed hippocampal neurons were immuno-stained for γ-actin (green), to visualize filopodia and spines and MAP2 (red) to highlight the dendritic arbor. Examples shown here are at 7 days in vitro (DIV) and 21 DIV. Arrows indicate examples of filopodia; arrowheads indicate examples of spines. The inset in the right-hand panel is an ∼ 3-fold enlargement of the boxed area. (B) Fixed hippocampal neurons were immuno-stained for PSD-95 (green), to visualize post-synaptic sites and synaptogamin (red) to highlight the pre-synaptic boutons. Overlapping puncta of PSD-95 and synaptotagmin (yellow) were considered to be synapses. Examples shown here are at 7 DIV and 21 DIV. Yellow arrowheads indicate examples of synapses. Arrows indicate examples or PSD-95 puncta without overlapping synaptotagmin puncta. Bar = 10 μm. (C) Graph illustrating the number of filopodia /100μm dendrite length comparing DIV 7, 14 and 21 hippocampal neurons (n=100 dendrites at least 100 μm long, from at least 25 separate neurons at DIV7, 14 or 21; 3 separate experiments). As the neurons mature, the density of filopodia declines. (D-F) Graphs illustrating the number of spines /100μm dendrite length (D), dendritic branches per neuron (E) and the number of synapses /100μm dendrite length (F), comparing DIV 7, 14 and 21 hippocampal neurons. In C, D and F, n=100 dendrites at least 100 μm long from at least 25 separate neurons at DIV 7, 14 or 21; 3 separate experiments. In E, n= at least 25 neurons at DIV 7, 14 or 21; 3 separate experiments). As the neurons mature, the density of synapses increases 15 fold, similar to the increase observed with spines, while branching of the dendritic arbor increases markedly. All data bars represent the mean ± SEM.

    Article Snippet: Primary antibodies used were mouse monoclonal antibody to PSD-95 (1:2000) (DakoCytomation, Inc., Carpinteria, CA), mouse monoclonal antibody to MAP2 (1:2000) (Chemicon International, Temecula, CA), rabbit antiserum to γ-actin (1:1000) (kindly provided by Dr. J. Bulinsky); rabbit antiserum to synaptotagmin I (V216; 1:1000) (kindly provided by Dr. T. Sudhof) and chicken monoclonal antibody to GFP (1:1500) (Aves Labs, Inc., Tigard, Or).

    Techniques: Staining, In Vitro

    Caveolin (Cav)-1 knock-out (KO) and Cav-3 KO mice have altered expression and membrane/lipid raft (MLR) localization of key neuronal and glial proteins. Sucrose density fractionation (SDF) and Western blot (WB) of wild-type (WT), Cav-1 KO, and Cav-3 KO brains. Buoyant fractions (BF) contain the cholesterol and sphingolipid enriched MLR, while heavy farctions (HF) contain non-MLR cellular components. (A) WB detection of PSD-95, NR2B, NR1 and TrkB expression in BF and HF. (B) WB detection of toll-like receptor-4 (TLR4), A A2 AR, A 3 AR, and A 1 AR expression in BF and HF. (C) WB detection of LRP-1 and LDL-R expression in BF and HF. Bottom left, WB analysis of GAPDH in whole cell lysates (WCL) from which SDF were generated for each group. Bottom right, WB shows loss of Cav-1 and Cav-3 protein expression in the transgenic mouse used in the present study.

    Journal: Journal of Neuroinflammation

    Article Title: Traumatic brain injury enhances neuroinflammation and lesion volume in caveolin deficient mice

    doi: 10.1186/1742-2094-11-39

    Figure Lengend Snippet: Caveolin (Cav)-1 knock-out (KO) and Cav-3 KO mice have altered expression and membrane/lipid raft (MLR) localization of key neuronal and glial proteins. Sucrose density fractionation (SDF) and Western blot (WB) of wild-type (WT), Cav-1 KO, and Cav-3 KO brains. Buoyant fractions (BF) contain the cholesterol and sphingolipid enriched MLR, while heavy farctions (HF) contain non-MLR cellular components. (A) WB detection of PSD-95, NR2B, NR1 and TrkB expression in BF and HF. (B) WB detection of toll-like receptor-4 (TLR4), A A2 AR, A 3 AR, and A 1 AR expression in BF and HF. (C) WB detection of LRP-1 and LDL-R expression in BF and HF. Bottom left, WB analysis of GAPDH in whole cell lysates (WCL) from which SDF were generated for each group. Bottom right, WB shows loss of Cav-1 and Cav-3 protein expression in the transgenic mouse used in the present study.

    Article Snippet: Reagents The following primary antibodies were used for Western blot (WB) and immunofluorescence microscopy (IF) analysis: Abcam (1 Kendall Square, Suite B2304, Cambridge, MA 02139-1517, USA) - A2A AR #ab79714, β3 -tubulin #ab11314, Cav-3 #ab2912, MAP2 #ab32454; BD Transduction Labs (2350 Qume Drive, San Jose, CA 95131, USA) - NR2B #610417, TrkB #610102; Cell Signaling (3 Trask Lane, Danvers, MA, 01923, USA) - AMPAR #2460 s, Cav-1 #3267, NR1 #4204, NR2A #4205, PSD-95 #2507; Epitomics (863 Mitten Road, Suite 103, Burlingame, CA, 94010-1303, USA) - LDLR #1956-1, LRP-1 #2703-1; Imgenex (11175 Flintkote Ave, Suite E, San Diego, CA, 92121, USA) - GAPDH #IMG-5019A-1; Millipore (290 Concord Road, Billerica, MA, 01821, USA) - GFAP AB5541; Santa Cruz (10410 Finnell Street, Dallas, TX, 75220, USA) - A1 AR sc-28995, A3 AR sc-12938, TLR4 sc-30002, goat anti-mouse IgG-HRP sc-2031, goat anti-rabbit IgG-HRP, sc-2030 goat anti-rat IgG-HRP sc-2006; Stressgen (4243 Glanford Avenue, Victoria, BC, Canada) - HSP90 #SPA835; WAKO (1-2 Doshomachi 3-Chome, Chuo-Ku, Osaka, 540-8605, Japan) - Iba1 WB #016-20001, IF #019-19741; Molecular Probes (3175 Staley Road, Grand Island, NY, 14072, USA) - goat anti-rabbit-488 IgG (H + L) #A11008, goat anti-mouse-594 IgG (H + L) #A11005.

    Techniques: Knock-Out, Mouse Assay, Expressing, Fractionation, Western Blot, Generated, Transgenic Assay

    Apo ER 2 exon 19 inclusion is lower in human and mouse AD brains compared to non‐ AD samples Schematic of Apo ER 2 expression and signaling via ligand binding (e.g., Reelin, ApoE). Apo ER 2 alternative splicing of exon 19 produces two Apo ER 2 protein isoforms with distinct functions. While both isoforms of Apo ER 2 (+ex19 and Δex19) can bind ligand, a protein domain of Apo ER 2 encoded by exon 19 interacts with PSD 95 to mediate association of Apo ER 2 with the NMDAR complex following Reelin binding. This signaling leads to calcium influx through NMDAR s and the induction of long‐term potentiation ( LTP ). RT – PCR analysis of RNA from mid‐temporal autopsy brain sections obtained from NCI , MCI , or AD subjects. Representative gel of results is shown. Quantitation of RT – PCR products of all samples analyzed from postmortem brain tissues (mean ± s.e.m.). An omnibus significant effect was obtained using the one‐way analysis of variance (one‐way ANOVA ) with Tukey posttest to determine the P ‐value. The number of samples analyzed ( n ) is indicated. RT – PCR analysis of RNA from the hippocampus of 1‐ to 2‐ and 3‐ to 7‐month‐old non‐transgenic ( WT ) or Tg CRND 8 ( AD ) mice. Quantification of the relative ratio of exon 19 inclusion for Tg CRND 8 ( AD ) and WT mice (mean ± s.e.m.; one‐way ANOVA with Tukey posttest). The number of samples analyzed ( n ) is indicated. Source data are available online for this figure.

    Journal: EMBO Molecular Medicine

    Article Title: Therapeutic correction of ApoER2 splicing in Alzheimer's disease mice using antisense oligonucleotides

    doi: 10.15252/emmm.201505846

    Figure Lengend Snippet: Apo ER 2 exon 19 inclusion is lower in human and mouse AD brains compared to non‐ AD samples Schematic of Apo ER 2 expression and signaling via ligand binding (e.g., Reelin, ApoE). Apo ER 2 alternative splicing of exon 19 produces two Apo ER 2 protein isoforms with distinct functions. While both isoforms of Apo ER 2 (+ex19 and Δex19) can bind ligand, a protein domain of Apo ER 2 encoded by exon 19 interacts with PSD 95 to mediate association of Apo ER 2 with the NMDAR complex following Reelin binding. This signaling leads to calcium influx through NMDAR s and the induction of long‐term potentiation ( LTP ). RT – PCR analysis of RNA from mid‐temporal autopsy brain sections obtained from NCI , MCI , or AD subjects. Representative gel of results is shown. Quantitation of RT – PCR products of all samples analyzed from postmortem brain tissues (mean ± s.e.m.). An omnibus significant effect was obtained using the one‐way analysis of variance (one‐way ANOVA ) with Tukey posttest to determine the P ‐value. The number of samples analyzed ( n ) is indicated. RT – PCR analysis of RNA from the hippocampus of 1‐ to 2‐ and 3‐ to 7‐month‐old non‐transgenic ( WT ) or Tg CRND 8 ( AD ) mice. Quantification of the relative ratio of exon 19 inclusion for Tg CRND 8 ( AD ) and WT mice (mean ± s.e.m.; one‐way ANOVA with Tukey posttest). The number of samples analyzed ( n ) is indicated. Source data are available online for this figure.

    Article Snippet: The ApoER2 protein domain encoded by this alternative exon mediates the interaction of ApoER2 with PSD‐95 to activate NMDA receptors and also with c‐Jun amino‐terminal kinase (JNK)‐interacting proteins (JIP), which have been implicated in neuronal survival through interactions with JNK (Gotthardt et al , ; Stockinger et al , ; Beffert et al , , ; Hoe et al , ).

    Techniques: Expressing, Ligand Binding Assay, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Quantitation Assay, Transgenic Assay, Mouse Assay

    Punctate and synaptic localisations of Vangl2 with or without PDZ-binding motif. ( a ) Rat hippocampal neurons were transfected with the indicated expression plasmids (upper: GFP-Vangl2 WT, middle: GFP-Vangl2 ΔETSV, lower: mGFP) and cultured for 21 days. GFP fluorescence (green), α-PSD-95 immunofluorescence (IF; red) as well as their merged images are presented. Note the punctate localisation of GFP-Vangl2 ΔETSV and the mostly flat signals of mGFP. ( b ) Bar graphs showing the density of the GFP puncta that were formed by the indicated expression constructs along the dendrites. Note the similar levels of puncta formation between GFP-Vangl2 WT and ΔETSV. ( c ) Bar graphs showing the ratio of the PSD-95-associated population of formed GFP puncta for each expression construct. Note that the clusters of Vangl2 ΔETSV are more associated with PSD-95 than those of the simply membrane-tethered protein (mGFP). The highly magnified images of the delimited region are shown in the respective lower panels. Some examples of the co-localised puncta are indicated with arrowheads. The data are presented as mean ± SD. Significant (p

    Journal: Scientific Reports

    Article Title: PDZ interaction of Vangl2 links PSD-95 and Prickle2 but plays only a limited role in the synaptic localisation of Vangl2

    doi: 10.1038/srep12916

    Figure Lengend Snippet: Punctate and synaptic localisations of Vangl2 with or without PDZ-binding motif. ( a ) Rat hippocampal neurons were transfected with the indicated expression plasmids (upper: GFP-Vangl2 WT, middle: GFP-Vangl2 ΔETSV, lower: mGFP) and cultured for 21 days. GFP fluorescence (green), α-PSD-95 immunofluorescence (IF; red) as well as their merged images are presented. Note the punctate localisation of GFP-Vangl2 ΔETSV and the mostly flat signals of mGFP. ( b ) Bar graphs showing the density of the GFP puncta that were formed by the indicated expression constructs along the dendrites. Note the similar levels of puncta formation between GFP-Vangl2 WT and ΔETSV. ( c ) Bar graphs showing the ratio of the PSD-95-associated population of formed GFP puncta for each expression construct. Note that the clusters of Vangl2 ΔETSV are more associated with PSD-95 than those of the simply membrane-tethered protein (mGFP). The highly magnified images of the delimited region are shown in the respective lower panels. Some examples of the co-localised puncta are indicated with arrowheads. The data are presented as mean ± SD. Significant (p

    Article Snippet: In the former situation, the tertiary protein complex that is formed between PSD-95, Vangl2 and Prickle2, which is presented in this study , may play a role in the establishment of the PSD.

    Techniques: Binding Assay, Transfection, Expressing, Cell Culture, Fluorescence, Immunofluorescence, Construct

    Interaction of Van Gogh-like 2 (Vangl2) with other postsynaptic density (PSD) proteins. ( a , b ) HEK293T cells were transfected with the indicated expression constructs, and the cell lysates as well as immuno-precipitates (IP) were analysed by western blotting (WB) with the indicated antibodies (Abs). IP was performed with either α-FLAG ( a ) or α-green fluorescent protein (GFP) ( b ) Abs. 0.5% of the cell lysate was loaded as Input. IB: immunoblot. ( c ) Bar graphs showing the precipitated ratio of Prickle2 in the WB analyses presented in ( b ). Note the robust enhancement of the complex formation between Prickle2 and PSD-95 by Vangl2 wild type (WT) but not by the ΔETSV. The amount of Prickle2 in the cell lysate was highly sensitive to the introduction of Vangl2 35 36 . The data are presented as mean ± standard deviation (SD). Significant differences (p

    Journal: Scientific Reports

    Article Title: PDZ interaction of Vangl2 links PSD-95 and Prickle2 but plays only a limited role in the synaptic localisation of Vangl2

    doi: 10.1038/srep12916

    Figure Lengend Snippet: Interaction of Van Gogh-like 2 (Vangl2) with other postsynaptic density (PSD) proteins. ( a , b ) HEK293T cells were transfected with the indicated expression constructs, and the cell lysates as well as immuno-precipitates (IP) were analysed by western blotting (WB) with the indicated antibodies (Abs). IP was performed with either α-FLAG ( a ) or α-green fluorescent protein (GFP) ( b ) Abs. 0.5% of the cell lysate was loaded as Input. IB: immunoblot. ( c ) Bar graphs showing the precipitated ratio of Prickle2 in the WB analyses presented in ( b ). Note the robust enhancement of the complex formation between Prickle2 and PSD-95 by Vangl2 wild type (WT) but not by the ΔETSV. The amount of Prickle2 in the cell lysate was highly sensitive to the introduction of Vangl2 35 36 . The data are presented as mean ± standard deviation (SD). Significant differences (p

    Article Snippet: In the former situation, the tertiary protein complex that is formed between PSD-95, Vangl2 and Prickle2, which is presented in this study , may play a role in the establishment of the PSD.

    Techniques: Transfection, Expressing, Construct, Western Blot, Standard Deviation

    Identification of the three types of Vangl2 clusters in terms of association with PSD-95. ( a ) The overall distribution of GFP-Vangl2 WT puncta on the dendrites of electroporated neurons in culture. An example of each type of punctum is indicate and marked with a capitalized letter (O: overlapped, C: complementary, U: unassociated). ( b , c , d ) The highly magnified images of the GFP signals of each GFP-Vangl2 WT punctum (green) merged with those of the IF signals of PSD-95 (red). Some examples of puncta with overlapped ( b ), highly associated but complementary ( c ) and essentially unassociated ( d ) localisation are presented. The puncta delimited in ( a ) are shown in the upper most row. ( e , f , g ) Plot-profile analysis of one of the merged images shown in ( b , c , d ). The images in the lowest row were analyzed. Note that the PSD-95 signals occupy the hollow of the GFP signals in this particular example ( f ). “O”s and the white lines shown in ( b ), ( c ) and ( d ) indicate the origins and tracks of the plot-profile analyses presented in ( e ), ( f ) and ( g ), respectively. Scale bars: 1 μm.

    Journal: Scientific Reports

    Article Title: PDZ interaction of Vangl2 links PSD-95 and Prickle2 but plays only a limited role in the synaptic localisation of Vangl2

    doi: 10.1038/srep12916

    Figure Lengend Snippet: Identification of the three types of Vangl2 clusters in terms of association with PSD-95. ( a ) The overall distribution of GFP-Vangl2 WT puncta on the dendrites of electroporated neurons in culture. An example of each type of punctum is indicate and marked with a capitalized letter (O: overlapped, C: complementary, U: unassociated). ( b , c , d ) The highly magnified images of the GFP signals of each GFP-Vangl2 WT punctum (green) merged with those of the IF signals of PSD-95 (red). Some examples of puncta with overlapped ( b ), highly associated but complementary ( c ) and essentially unassociated ( d ) localisation are presented. The puncta delimited in ( a ) are shown in the upper most row. ( e , f , g ) Plot-profile analysis of one of the merged images shown in ( b , c , d ). The images in the lowest row were analyzed. Note that the PSD-95 signals occupy the hollow of the GFP signals in this particular example ( f ). “O”s and the white lines shown in ( b ), ( c ) and ( d ) indicate the origins and tracks of the plot-profile analyses presented in ( e ), ( f ) and ( g ), respectively. Scale bars: 1 μm.

    Article Snippet: In the former situation, the tertiary protein complex that is formed between PSD-95, Vangl2 and Prickle2, which is presented in this study , may play a role in the establishment of the PSD.

    Techniques:

    Statistical analysis of the synaptic localisations of Vangl2 with or without PDZ-binding motif. ( a ) Overall distribution of GFP-Vangl2 ΔETSV and PSD-95 puncta on the dendrites of electroporated neurons in culture. An example of each type of punctum is indicated and marked with a capitalized letter (O: overlapped, C: complementary, U: unassociated). ( b ) The highly magnified images of the indicated regions in ( a ), showing the overlapped, complementary and unassociated localisation with PSD-95 IF. ( c ) Bar graphs showing the ratio of the GFP puncta with overlapped co-localisation with PSD-95. Note the significant reduction in the overlapped ratio by the deletion of PBM (ΔETSV). ( d ) Bar graphs showing the ratio of GFP puncta with a complementary pattern of association with PSD-95. Note that the deletion of the PBM did not affect the occurrence of the complementary association. ( e ) Bar graphs showing the ratio of GFP puncta without association with PSD-95. Scale bars: 20 μm ( a ) and 5 μm ( b ). The data are presented as mean ± SD. Significant (p

    Journal: Scientific Reports

    Article Title: PDZ interaction of Vangl2 links PSD-95 and Prickle2 but plays only a limited role in the synaptic localisation of Vangl2

    doi: 10.1038/srep12916

    Figure Lengend Snippet: Statistical analysis of the synaptic localisations of Vangl2 with or without PDZ-binding motif. ( a ) Overall distribution of GFP-Vangl2 ΔETSV and PSD-95 puncta on the dendrites of electroporated neurons in culture. An example of each type of punctum is indicated and marked with a capitalized letter (O: overlapped, C: complementary, U: unassociated). ( b ) The highly magnified images of the indicated regions in ( a ), showing the overlapped, complementary and unassociated localisation with PSD-95 IF. ( c ) Bar graphs showing the ratio of the GFP puncta with overlapped co-localisation with PSD-95. Note the significant reduction in the overlapped ratio by the deletion of PBM (ΔETSV). ( d ) Bar graphs showing the ratio of GFP puncta with a complementary pattern of association with PSD-95. Note that the deletion of the PBM did not affect the occurrence of the complementary association. ( e ) Bar graphs showing the ratio of GFP puncta without association with PSD-95. Scale bars: 20 μm ( a ) and 5 μm ( b ). The data are presented as mean ± SD. Significant (p

    Article Snippet: In the former situation, the tertiary protein complex that is formed between PSD-95, Vangl2 and Prickle2, which is presented in this study , may play a role in the establishment of the PSD.

    Techniques: Binding Assay

    Immunoblot analysis of expression ratios of the pre and postsynaptic proteins in Aβ-GFP Tg and non-Tg mice synapse. ( A,B ) Synaptosome fractions from hippocampus (20 μg/lane) were immunoblotted against presynaptic markers ( A ; synaptophysin, VAMP-2, syntaxin-1, and Munc13–1) and postsynaptic markers ( B ; PSD-95, GluN1, GluN2A, GluN2B, GluA2, and neuroligin). We prepared 4 sets of Aβ-GFP Tg/non-Tg samples. Band intensities of Aβ-GFP Tg mice were normalized by those of actin and the expression were calculated ratios with the values of non-Tg mice as 1. There was no difference in expression ratios of the presynaptic proteins we examined. However, expression of the postsynaptic proteins GluN2B, Glu2A, and neuroligin were significantly decreased in Aβ-GFP Tg mice at 3-month-old (*p

    Journal: Scientific Reports

    Article Title: New Alzheimer’s disease model mouse specialized for analyzing the function and toxicity of intraneuronal Amyloid β oligomers

    doi: 10.1038/s41598-019-53415-8

    Figure Lengend Snippet: Immunoblot analysis of expression ratios of the pre and postsynaptic proteins in Aβ-GFP Tg and non-Tg mice synapse. ( A,B ) Synaptosome fractions from hippocampus (20 μg/lane) were immunoblotted against presynaptic markers ( A ; synaptophysin, VAMP-2, syntaxin-1, and Munc13–1) and postsynaptic markers ( B ; PSD-95, GluN1, GluN2A, GluN2B, GluA2, and neuroligin). We prepared 4 sets of Aβ-GFP Tg/non-Tg samples. Band intensities of Aβ-GFP Tg mice were normalized by those of actin and the expression were calculated ratios with the values of non-Tg mice as 1. There was no difference in expression ratios of the presynaptic proteins we examined. However, expression of the postsynaptic proteins GluN2B, Glu2A, and neuroligin were significantly decreased in Aβ-GFP Tg mice at 3-month-old (*p

    Article Snippet: VAMP-2 (1:5000, Abcam, Cambridge, UK), Munc13-1(1:1000, Synaptic Systems, Gottingen, Germany), syntaxin-1 (1:1000, Merck), synaptophysin (1:2000, Sigma-Aldrich), PSD-95 (1:200, Merck), GluN1 (1:200, Merck), GluN2A (1:500, Merck), GluN2B (1:200, Merck), GluA2 (1:200, Merck), neuroligin 1(1:500, Synaptic Systems), and actin (1:2000, Merck) antibodies were used to probe the corresponding proteins and ECL was used for detection (Immunostar Zeta and Immunostar LD, FUJIFILM Wako Pure Chemical Corporation).

    Techniques: Expressing, Mouse Assay

    Postsynaptic receptor scaffolding proteins and subunits. In contralateral V1, PSD-95 ( A ) and gephyrin ( B ) had a similar pattern of changes: fluoxetine alone had no effect, MD alone caused a loss of expression, but combining fluoxetine with MD prevented the MD-induced loss and caused super-compensation above normal levels. GluN1 ( C ) was reduced by fluoxetine regardless of visual experience, whereas MD alone caused an increase. GluA2 ( D ) was unaffected by fluoxetine alone, MD caused an increase, but combing fluoxetine with MD caused a decrease. GluN2B ( E ) was reduced by fluoxetine regardless of visual experience, whereas MD had no effect. GluN2A ( F ) expression of each experimental group was not different from normal animals, but the MDed group had higher expression than either fluoxetine alone or fluoxetine combined with MD. GABAAα3 ( G ) was unaffected by fluoxetine alone, MD caused an increase, but combing fluoxetine with MD prevented the MD-induced increase. GABAAα1 ( H ) was increased by fluoxetine regardless of visual experience, while MD alone had no effect. * p

    Journal: eNeuro

    Article Title: Effects of Fluoxetine and Visual Experience on Glutamatergic and GABAergic Synaptic Proteins in Adult Rat Visual Cortex of Fluoxetine and Visual Experience on Glutamatergic and GABAergic Synaptic Proteins in Adult Rat Visual Cortex of Fluoxetine and Visual Experience on Glutamatergic and GABAergic Synaptic Proteins in Adult Rat Visual Cortex

    doi: 10.1523/ENEURO.0126-15.2015

    Figure Lengend Snippet: Postsynaptic receptor scaffolding proteins and subunits. In contralateral V1, PSD-95 ( A ) and gephyrin ( B ) had a similar pattern of changes: fluoxetine alone had no effect, MD alone caused a loss of expression, but combining fluoxetine with MD prevented the MD-induced loss and caused super-compensation above normal levels. GluN1 ( C ) was reduced by fluoxetine regardless of visual experience, whereas MD alone caused an increase. GluA2 ( D ) was unaffected by fluoxetine alone, MD caused an increase, but combing fluoxetine with MD caused a decrease. GluN2B ( E ) was reduced by fluoxetine regardless of visual experience, whereas MD had no effect. GluN2A ( F ) expression of each experimental group was not different from normal animals, but the MDed group had higher expression than either fluoxetine alone or fluoxetine combined with MD. GABAAα3 ( G ) was unaffected by fluoxetine alone, MD caused an increase, but combing fluoxetine with MD prevented the MD-induced increase. GABAAα1 ( H ) was increased by fluoxetine regardless of visual experience, while MD alone had no effect. * p

    Article Snippet: We began by examining expression of synapsin, synaptophysin, PSD-95, and gephyrin in V1 ipsilateral to the deprived eye.

    Techniques: Scaffolding, Expressing

    Presynaptic and postsynaptic proteins in ipsilateral V1. In V1 ipsilateral to the deprived eye, there was no effect of experimental condition on the expression of synapsin ( A ), synaptophysin ( B ), PSD-95 ( C ), or gephyrin ( D ). * p

    Journal: eNeuro

    Article Title: Effects of Fluoxetine and Visual Experience on Glutamatergic and GABAergic Synaptic Proteins in Adult Rat Visual Cortex of Fluoxetine and Visual Experience on Glutamatergic and GABAergic Synaptic Proteins in Adult Rat Visual Cortex of Fluoxetine and Visual Experience on Glutamatergic and GABAergic Synaptic Proteins in Adult Rat Visual Cortex

    doi: 10.1523/ENEURO.0126-15.2015

    Figure Lengend Snippet: Presynaptic and postsynaptic proteins in ipsilateral V1. In V1 ipsilateral to the deprived eye, there was no effect of experimental condition on the expression of synapsin ( A ), synaptophysin ( B ), PSD-95 ( C ), or gephyrin ( D ). * p

    Article Snippet: We began by examining expression of synapsin, synaptophysin, PSD-95, and gephyrin in V1 ipsilateral to the deprived eye.

    Techniques: Expressing

    S561A mutation facilitates the interaction between PSD-95 and its synaptic binding partners. a , GST pulldown assay for GKAP. GST or GST-SH3-GK WT/S561A beads were incubated with whole-brain lysates. Bound proteins were analyzed by Western blotting with the GKAP antibody. b , quantification of blots in a . The error bar in the scatter plot represents S.D. n = 4. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Postsynaptic density 95 (PSD-95) serine 561 phosphorylation regulates a conformational switch and bidirectional dendritic spine structural plasticity

    doi: 10.1074/jbc.M117.782490

    Figure Lengend Snippet: S561A mutation facilitates the interaction between PSD-95 and its synaptic binding partners. a , GST pulldown assay for GKAP. GST or GST-SH3-GK WT/S561A beads were incubated with whole-brain lysates. Bound proteins were analyzed by Western blotting with the GKAP antibody. b , quantification of blots in a . The error bar in the scatter plot represents S.D. n = 4. *, p

    Article Snippet: Nelson C. D., Kim M. J., Hsin H., Chen Y., and Sheng M. (2013) Phosphorylation of threonine-19 of PSD-95 by GSK-3β is required for PSD-95 mobilization and long-term depression .

    Techniques: Mutagenesis, Binding Assay, GST Pulldown Assay, Incubation, Western Blot

    Effects of Ser-561 phosphorylation mutants of PSD-95 on SH3-GK interaction. a , a schematic representation of the structure of PSD-95 and the single-chain FRET probes (CFP-SH3-GK-YFP with or without mutation). b , GST pulldown assay. HEK293 cells expressing GFP-GK or GFP-GK S561A/D were incubated with GST-SH3 beads. GST beads were used as a control. The bound proteins were analyzed by Western blotting ( WB ) with a GFP antibody. c , quantification for the GST pulldown assay revealed that significantly less GFP-GK S561A was pulled down with GST-SH3 as compared with wild-type GFP-GK, whereas more GFP-GK S561D was pulled down as compared with the wild-type counterpart. Error bars in the scatter plot represent S.D. n = 3 independent experiments. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Postsynaptic density 95 (PSD-95) serine 561 phosphorylation regulates a conformational switch and bidirectional dendritic spine structural plasticity

    doi: 10.1074/jbc.M117.782490

    Figure Lengend Snippet: Effects of Ser-561 phosphorylation mutants of PSD-95 on SH3-GK interaction. a , a schematic representation of the structure of PSD-95 and the single-chain FRET probes (CFP-SH3-GK-YFP with or without mutation). b , GST pulldown assay. HEK293 cells expressing GFP-GK or GFP-GK S561A/D were incubated with GST-SH3 beads. GST beads were used as a control. The bound proteins were analyzed by Western blotting ( WB ) with a GFP antibody. c , quantification for the GST pulldown assay revealed that significantly less GFP-GK S561A was pulled down with GST-SH3 as compared with wild-type GFP-GK, whereas more GFP-GK S561D was pulled down as compared with the wild-type counterpart. Error bars in the scatter plot represent S.D. n = 3 independent experiments. *, p

    Article Snippet: Nelson C. D., Kim M. J., Hsin H., Chen Y., and Sheng M. (2013) Phosphorylation of threonine-19 of PSD-95 by GSK-3β is required for PSD-95 mobilization and long-term depression .

    Techniques: Mutagenesis, GST Pulldown Assay, Expressing, Incubation, Western Blot

    Effects of Ser-561 phosphorylation mutants on PSD-95 dynamics at the synapse. a , representative time-lapse FRAP images of PSD-95-GFP constructs in DIV24 hippocampal neurons. PSD-95 clusters at the spine head ( yellow arrow ) were bleached using a 488 nm laser set at 100% laser power, and the fluorescence recovery was followed over time. Scale bar , 2 μm. b , quantification of fluorescence recovery. Error bars represent S.D. n = 30–35 spines from 16 hippocampal neurons per group. **, p

    Journal: The Journal of Biological Chemistry

    Article Title: Postsynaptic density 95 (PSD-95) serine 561 phosphorylation regulates a conformational switch and bidirectional dendritic spine structural plasticity

    doi: 10.1074/jbc.M117.782490

    Figure Lengend Snippet: Effects of Ser-561 phosphorylation mutants on PSD-95 dynamics at the synapse. a , representative time-lapse FRAP images of PSD-95-GFP constructs in DIV24 hippocampal neurons. PSD-95 clusters at the spine head ( yellow arrow ) were bleached using a 488 nm laser set at 100% laser power, and the fluorescence recovery was followed over time. Scale bar , 2 μm. b , quantification of fluorescence recovery. Error bars represent S.D. n = 30–35 spines from 16 hippocampal neurons per group. **, p

    Article Snippet: Nelson C. D., Kim M. J., Hsin H., Chen Y., and Sheng M. (2013) Phosphorylation of threonine-19 of PSD-95 by GSK-3β is required for PSD-95 mobilization and long-term depression .

    Techniques: Construct, Fluorescence

    NR1 and PSD-95 proteins in PSD-enriched fractions from prefrontal cortex are reduced in people with schizophrenia. NR1 protein levels, normalized to β-III-tubulin, were quantified by western blot images captured by Chemidoc (representative blot captured on film shown in a ). Single bands for NR1 and β-III-tubulin were present at the predicted sizes (~120 and 50 kDa, respectively). NR1 protein levels (average of three replicates) were decreased compared to matched controls ( c ). PSD-95 protein levels, normalized to actin, were quantified by western blot images captured on film (representative blot captured on film shown in b ). Single bands for PSD-95 and actin were present in the predicted sizes (~105 and 42 kDa, respectively). PSD-95 protein levels (average of two replicates) were decreased compared with matched controls ( d ). ** P

    Journal: NPJ Schizophrenia

    Article Title: Postsynaptic density levels of the NMDA receptor NR1 subunit and PSD-95 protein in prefrontal cortex from people with schizophrenia

    doi: 10.1038/npjschz.2015.37

    Figure Lengend Snippet: NR1 and PSD-95 proteins in PSD-enriched fractions from prefrontal cortex are reduced in people with schizophrenia. NR1 protein levels, normalized to β-III-tubulin, were quantified by western blot images captured by Chemidoc (representative blot captured on film shown in a ). Single bands for NR1 and β-III-tubulin were present at the predicted sizes (~120 and 50 kDa, respectively). NR1 protein levels (average of three replicates) were decreased compared to matched controls ( c ). PSD-95 protein levels, normalized to actin, were quantified by western blot images captured on film (representative blot captured on film shown in b ). Single bands for PSD-95 and actin were present in the predicted sizes (~105 and 42 kDa, respectively). PSD-95 protein levels (average of two replicates) were decreased compared with matched controls ( d ). ** P

    Article Snippet: The membranes were probed with primary antibodies for SNAP-25 (1:2,500 dilution, NCL-SNAP-25, Novocastra, Leica Biosystems, Noth Ryde, NSW, Australia), synaptobrevin (1:3,000 dilution, MAB 373, Chemicon, Merck Millipore, Sydney, NSW, Australia), calnexin (1:1,000, Cat # 2433, Cell Signaling, Merck Millipore) PSD-95 ( ) and α-synaptophysin (1:5,000 dilution, MAB329, Merck Millipore) followed by horseradish peroxidase conjugated secondary antibody (1:5,000 dilution, anti-mouse AP124P or anti-rabbit AP188P as appropriate, Merck Millipore).

    Techniques: Western Blot

    PSD-95 in tissue fractions from prefrontal cortex. Successful enrichment of the postsynaptic density (PSD) is indicated by the presence of increasing concentrations of PSD-95 in 1.5 μg of ‘total fraction’ (T), ‘synaptic membrane fraction’ (SPM) and ‘PSD-enriched fraction’ pooled samples ( a and b ). Quantification of band densities suggests greater enrichment of PSD-95 in control than in schizophrenia PSD-enriched samples ( c ). C, cytosolic fraction; SV, synaptic vesicle fraction.

    Journal: NPJ Schizophrenia

    Article Title: Postsynaptic density levels of the NMDA receptor NR1 subunit and PSD-95 protein in prefrontal cortex from people with schizophrenia

    doi: 10.1038/npjschz.2015.37

    Figure Lengend Snippet: PSD-95 in tissue fractions from prefrontal cortex. Successful enrichment of the postsynaptic density (PSD) is indicated by the presence of increasing concentrations of PSD-95 in 1.5 μg of ‘total fraction’ (T), ‘synaptic membrane fraction’ (SPM) and ‘PSD-enriched fraction’ pooled samples ( a and b ). Quantification of band densities suggests greater enrichment of PSD-95 in control than in schizophrenia PSD-enriched samples ( c ). C, cytosolic fraction; SV, synaptic vesicle fraction.

    Article Snippet: The membranes were probed with primary antibodies for SNAP-25 (1:2,500 dilution, NCL-SNAP-25, Novocastra, Leica Biosystems, Noth Ryde, NSW, Australia), synaptobrevin (1:3,000 dilution, MAB 373, Chemicon, Merck Millipore, Sydney, NSW, Australia), calnexin (1:1,000, Cat # 2433, Cell Signaling, Merck Millipore) PSD-95 ( ) and α-synaptophysin (1:5,000 dilution, MAB329, Merck Millipore) followed by horseradish peroxidase conjugated secondary antibody (1:5,000 dilution, anti-mouse AP124P or anti-rabbit AP188P as appropriate, Merck Millipore).

    Techniques:

    PSD-95 deletion impairs retrieval and stability of remote fear memories ( a ) Schematic of experimental design for testing remote retrieval of a cued fear memory. ( b ) PSD-95 GK mice froze less than WT controls during remote fear memory retrieval (n=8–9). ( c ) Schematic of experimental design for testing the remote retrieval of a contextual fear memory. ( d ) PSD-95 GK mice froze less than WT controls during remote fear memory retrieval (n=9–10). ( e ) Schematic of experimental design for testing recent and remote retrieval of a contextual fear memory in the same mice. ( f ) PSD-95 GK mice froze less than WT controls during remote, but not recent, fear memory retrieval (n=8). Data are means ±SEM. * P

    Journal: Molecular psychiatry

    Article Title: Durable fear memories require PSD-95

    doi: 10.1038/mp.2014.161

    Figure Lengend Snippet: PSD-95 deletion impairs retrieval and stability of remote fear memories ( a ) Schematic of experimental design for testing remote retrieval of a cued fear memory. ( b ) PSD-95 GK mice froze less than WT controls during remote fear memory retrieval (n=8–9). ( c ) Schematic of experimental design for testing the remote retrieval of a contextual fear memory. ( d ) PSD-95 GK mice froze less than WT controls during remote fear memory retrieval (n=9–10). ( e ) Schematic of experimental design for testing recent and remote retrieval of a contextual fear memory in the same mice. ( f ) PSD-95 GK mice froze less than WT controls during remote, but not recent, fear memory retrieval (n=8). Data are means ±SEM. * P

    Article Snippet: Extinction and precision of recent fear memories To further probe the nature of the fear memories formed in the absence of PSD-95, we examined fear extinction and memory precision.

    Techniques: Mouse Assay

    PSD-95 knockdown in infralimbic cortex impairs remote fear memory retrieval ( a ) Example of green fluorescent protein (GFP) localization of the PSD-95 knockdown adenoassociated virus in the IL (scale bar=200 µm). ( b ) Example Western blots showing viral-induced knockdown of PSD-95 protein. ( c ) Schematic of experimental design for testing recent and remote retrieval of a cued fear memory. ( d ) IL PSD-95 KD mice showed less freezing than GFP-virus controls during remote, but not recent retrieval (n=12–17). ( e ) ACC PSD-95 KD mice showed similar freezing levels to GFP controls during recent and remote retrieval (n=9–11). Inset: example of GFP localization of the PSD-95 KD adenoassociated virus in the Cg1 region of the ACC (scale bar=200 µm). ( f ) Schematic of experimental design for testing fear extinction. ( g ) IL PSD-95 KD mice decreased freezing from the first to the last extinction trial-block, but less so than GFP-virus controls. GFP-virus controls, but not IL PSD-95 KD mice, froze less during extinction retrieval as compared to the first trial-block of extinction training (n=7–9). ( h ) Example images of dendritic spines in IL pyramidal neurons from a PSD-95 KD (top) and GFP control (bottom) (scale bar=15 µm). ( i ) Examples and cartoon showing examples of narrow (N) and wide (W) subpopulations of dendritic spines. ( j,k ) PSD-95 KD decreased the spine head width, but not density, of relatively wide spines on IL pyramidal neurons, relative to GFP controls (n=7 mice per group, n=15–23 cells per group). Data are means ±SEM. * P

    Journal: Molecular psychiatry

    Article Title: Durable fear memories require PSD-95

    doi: 10.1038/mp.2014.161

    Figure Lengend Snippet: PSD-95 knockdown in infralimbic cortex impairs remote fear memory retrieval ( a ) Example of green fluorescent protein (GFP) localization of the PSD-95 knockdown adenoassociated virus in the IL (scale bar=200 µm). ( b ) Example Western blots showing viral-induced knockdown of PSD-95 protein. ( c ) Schematic of experimental design for testing recent and remote retrieval of a cued fear memory. ( d ) IL PSD-95 KD mice showed less freezing than GFP-virus controls during remote, but not recent retrieval (n=12–17). ( e ) ACC PSD-95 KD mice showed similar freezing levels to GFP controls during recent and remote retrieval (n=9–11). Inset: example of GFP localization of the PSD-95 KD adenoassociated virus in the Cg1 region of the ACC (scale bar=200 µm). ( f ) Schematic of experimental design for testing fear extinction. ( g ) IL PSD-95 KD mice decreased freezing from the first to the last extinction trial-block, but less so than GFP-virus controls. GFP-virus controls, but not IL PSD-95 KD mice, froze less during extinction retrieval as compared to the first trial-block of extinction training (n=7–9). ( h ) Example images of dendritic spines in IL pyramidal neurons from a PSD-95 KD (top) and GFP control (bottom) (scale bar=15 µm). ( i ) Examples and cartoon showing examples of narrow (N) and wide (W) subpopulations of dendritic spines. ( j,k ) PSD-95 KD decreased the spine head width, but not density, of relatively wide spines on IL pyramidal neurons, relative to GFP controls (n=7 mice per group, n=15–23 cells per group). Data are means ±SEM. * P

    Article Snippet: Extinction and precision of recent fear memories To further probe the nature of the fear memories formed in the absence of PSD-95, we examined fear extinction and memory precision.

    Techniques: Western Blot, Mouse Assay, Blocking Assay

    PSD-95 deletion does not affect retrieval of recent fear memories, but impairs their extinction and precision ( a ) Schematic of experimental design for testing recent fear memory retrieval following delay conditioning. ( b ) Genotypes showed equivalent levels of freezing during cue and context retrieval (n=11–17). ( c ) Schematic of experimental design for testing recent fear memory retrieval following trace conditioning. ( d ) Following trace fear conditioning, genotypes showed similar cue fear retrieval, but PSD-95 GK mice froze significantly less than wild-type controls during context retrieval (n=8–10). ( e ) Schematic of experimental design for testing extinction of a recent fear memory. ( f ) WT controls, but not PSD-95 GK mice, decreased freezing from the first to the last extinction trial-block, and froze less during extinction retrieval as compared to the first trial-block of extinction training (n=11). ( g ) Schematic of experimental design for testing the precision of a recent contextual fear memory. ( h ) WT controls, but not PSD-95 GK mice, froze more to the conditioned context than either of two different contexts (n=13). Data are means ± SEM. * P

    Journal: Molecular psychiatry

    Article Title: Durable fear memories require PSD-95

    doi: 10.1038/mp.2014.161

    Figure Lengend Snippet: PSD-95 deletion does not affect retrieval of recent fear memories, but impairs their extinction and precision ( a ) Schematic of experimental design for testing recent fear memory retrieval following delay conditioning. ( b ) Genotypes showed equivalent levels of freezing during cue and context retrieval (n=11–17). ( c ) Schematic of experimental design for testing recent fear memory retrieval following trace conditioning. ( d ) Following trace fear conditioning, genotypes showed similar cue fear retrieval, but PSD-95 GK mice froze significantly less than wild-type controls during context retrieval (n=8–10). ( e ) Schematic of experimental design for testing extinction of a recent fear memory. ( f ) WT controls, but not PSD-95 GK mice, decreased freezing from the first to the last extinction trial-block, and froze less during extinction retrieval as compared to the first trial-block of extinction training (n=11). ( g ) Schematic of experimental design for testing the precision of a recent contextual fear memory. ( h ) WT controls, but not PSD-95 GK mice, froze more to the conditioned context than either of two different contexts (n=13). Data are means ± SEM. * P

    Article Snippet: Extinction and precision of recent fear memories To further probe the nature of the fear memories formed in the absence of PSD-95, we examined fear extinction and memory precision.

    Techniques: Mouse Assay, Blocking Assay

    PSD-95 deletion disrupts activation of the infralimbic cortex during remote fear memory retrieval ( a ) Schematic of experimental design for testing recent and remote retrieval of a cued fear memory. ( b ) PSD-95 GK mice froze less than WT controls during remote, but not recent, fear memory retrieval. ( c ) Representative Zif268-labelled coronal section showing the three prefrontal regions analyzed. ( d ) IEG analysis of prefrontal regions found no change in the number of Zif268-positive cells in either the anterior cingulate cortex (ACC, Cg1 subregion) or prelimbic cortex (PL), irrespective of memory age or genotype. ( e ) WT mice exhibited an elevated number of Zif268-positive cells in the infralimbic cortex (IL) after remote, as compared to recent retrieval, while the number of cells did not increase in the PSD-95 GK mice. ( f ) Representative coronal sections showing Zif268-positive cells in the IL (scale bar= 150 µm). ( g ) Example images of dendritic spines in IL pyramidal neurons from a PSD-95 KO (top) and WT controls (bottom) (scale bar=15 µm). ( h ) PSD-95 KO mice had marginally lesser density and significantly lesser spine head width on IL pyramidal neurons, relative to WT controls (n=3 mice per group, n=7–12 cells per group). Data are means ±SEM. * P

    Journal: Molecular psychiatry

    Article Title: Durable fear memories require PSD-95

    doi: 10.1038/mp.2014.161

    Figure Lengend Snippet: PSD-95 deletion disrupts activation of the infralimbic cortex during remote fear memory retrieval ( a ) Schematic of experimental design for testing recent and remote retrieval of a cued fear memory. ( b ) PSD-95 GK mice froze less than WT controls during remote, but not recent, fear memory retrieval. ( c ) Representative Zif268-labelled coronal section showing the three prefrontal regions analyzed. ( d ) IEG analysis of prefrontal regions found no change in the number of Zif268-positive cells in either the anterior cingulate cortex (ACC, Cg1 subregion) or prelimbic cortex (PL), irrespective of memory age or genotype. ( e ) WT mice exhibited an elevated number of Zif268-positive cells in the infralimbic cortex (IL) after remote, as compared to recent retrieval, while the number of cells did not increase in the PSD-95 GK mice. ( f ) Representative coronal sections showing Zif268-positive cells in the IL (scale bar= 150 µm). ( g ) Example images of dendritic spines in IL pyramidal neurons from a PSD-95 KO (top) and WT controls (bottom) (scale bar=15 µm). ( h ) PSD-95 KO mice had marginally lesser density and significantly lesser spine head width on IL pyramidal neurons, relative to WT controls (n=3 mice per group, n=7–12 cells per group). Data are means ±SEM. * P

    Article Snippet: Extinction and precision of recent fear memories To further probe the nature of the fear memories formed in the absence of PSD-95, we examined fear extinction and memory precision.

    Techniques: Activation Assay, Mouse Assay

    PSD-95 deletion disrupts CS-related single-unit activity in the infralimbic cortex during fear memory retrieval ( a ) PSD-95 GK mice showed less CS-related IL single-unit activity than WT controls during recent (left panel) and remote (right panel) fear memory retrieval. Insets show first 100 millisecond timebins. ( b ) PSD-95 GK mice had fewer phasic CS-related IL single-units than WT controls during recent and remote retrieval. ( c ) IL single-unit activity correlated positively with freezing during remote retrieval. ( d ) IL single-unit burst activity increased during the CS in both genotypes during recent retrieval, but only in WT mice during remote retrieval. ( e ) PSD-95 GK mice showed lower gamma and theta oscillations than WT controls, on recent and remote retrievals. ( f ) Representative perievent spectrograms showing the gamma and theta frequency spectra around CS-onset. For gamma panels, darkest blue=0 mV 2 *s and darkest red=2e −5 . For theta panels, darkest blue=0 and darkest red=4e −4 mV 2 *s. Data are means ±SEM. n=6 mice per genotype, n=74–87 single-units per genotype. * P

    Journal: Molecular psychiatry

    Article Title: Durable fear memories require PSD-95

    doi: 10.1038/mp.2014.161

    Figure Lengend Snippet: PSD-95 deletion disrupts CS-related single-unit activity in the infralimbic cortex during fear memory retrieval ( a ) PSD-95 GK mice showed less CS-related IL single-unit activity than WT controls during recent (left panel) and remote (right panel) fear memory retrieval. Insets show first 100 millisecond timebins. ( b ) PSD-95 GK mice had fewer phasic CS-related IL single-units than WT controls during recent and remote retrieval. ( c ) IL single-unit activity correlated positively with freezing during remote retrieval. ( d ) IL single-unit burst activity increased during the CS in both genotypes during recent retrieval, but only in WT mice during remote retrieval. ( e ) PSD-95 GK mice showed lower gamma and theta oscillations than WT controls, on recent and remote retrievals. ( f ) Representative perievent spectrograms showing the gamma and theta frequency spectra around CS-onset. For gamma panels, darkest blue=0 mV 2 *s and darkest red=2e −5 . For theta panels, darkest blue=0 and darkest red=4e −4 mV 2 *s. Data are means ±SEM. n=6 mice per genotype, n=74–87 single-units per genotype. * P

    Article Snippet: Extinction and precision of recent fear memories To further probe the nature of the fear memories formed in the absence of PSD-95, we examined fear extinction and memory precision.

    Techniques: Activity Assay, Mouse Assay