Journal: International journal of molecular sciences

Article Title: Daily Brief Heat Therapy Reduces Seizures in A350V IQSEC2 Mice and Is Associated with Correction of AMPA Receptor-Mediated Synaptic Dysfunction.

doi: 10.3390/ijms24043924

Figure Lengend Snippet: Figure 4. Surface GluA2 AMPA receptor in neonatal hippocampal neurons is decreased in A350V neurons and is increased after heat treatment. (A) Representative images (20× magnification) of total (T.GluA2) and surface (S.GluA2) GluA2 AMPA receptor in wild-type (WT) and mutant (A350V) IQSEC2 hippocampal neurons. Neurons treated with heat (40 ◦C) for 1 h are labelled as MUT.H.S or WT.H.S. (B) Quantification of relative surface GluA2 expression (ratio: S.GluA2/T.GluA2). Data are reported as the mean ± SEM obtained from at least 4 neuronal culture preparations for each of the 4 groups. ** p < 0.01, *** p < 0.001, n.s. p > 0.05.

Article Snippet: To label neuronal surface GluA2 AMPAR, coverslips were incubated at 4 ◦C overnight in PBS containing 3% normal goat serum and mouse monoclonal extracellular anti-N-terminal GluA2 IgG (Synaptic System, Gottingen, Germany, cat# 182111).

Techniques: Mutagenesis, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: GRIP1 regulates synaptic plasticity and learning and memory

doi: 10.1073/pnas.2014827117

Figure Lengend Snippet: GRIP1 is recruited into synapses during cLTP. (A) Representative Western blots of proteins from PSD and total cell lysates (total) isolated from rat cortical neurons treated with (+) or without (−) glycine. (B) Quantification of protein levels in PSD (n = 5; Mann–Whitney U test). (C) Quantification of protein levels in total cell lysates. (n = 8; Student’s t test). (D) Representative Western blots of GRIP1 in P2 and S2 fractions isolated from rat cortical neurons treated with (+) or without (−) glycine. (E) Quantification of GRIP1 protein level in each fraction following cLTP (n = 15 to 16; Mann–Whitney U test). (F) Model of GRIP1 translocation during cLTP. Data are presented as mean ± SEM. n.s., not significant. **P < 0.01.

Article Snippet: The following antibodies were used: anti–beta-tubulin mAb (Sigma-Aldrich), anti-GluA1 N-terminal antibody mAb (4.9D, made in-house), anti-GluA2 N-terminal antibody mAb (032.19.9, made in-house), anti-GluA2 phospho-S880 specific mAb (02.22.4, made in-house), anti-GluA2 phospho-Y876 specific mAb (045.10.5, made in-house), anti-PSD95 mAb (NeuroMab), anti-GluA3 pAb (JH4300, made in-house), anti-GRIP1 mAb (BD Biosciences), anti-GRIP1 pAb (Chemicon), and anti-GRIP1 pAb (JH2260, made in-house).

Techniques: Western Blot, Isolation, MANN-WHITNEY, Translocation Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: GRIP1 regulates synaptic plasticity and learning and memory

doi: 10.1073/pnas.2014827117

Figure Lengend Snippet: Activity-dependent up-regulation of synaptic AMPARs requires GRIP1 expression. (A) Representative Western blots of proteins from PSD and total cell lysates from WT or Grip1 KO mouse neurons treated with (+) or without (−) glycine. (B) Quantification of protein levels in PSD under basal conditions in WT and Grip1 KO mouse neurons (n = 9 to 11; Student’s t test). (C) Quantification of protein levels in PSD from WT or Grip1 KO mouse neurons treated with (+) or without (−) glycine (n = 15; Mann–Whitney U test). Data are presented as mean ± SEM. n.s., not significant. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The following antibodies were used: anti–beta-tubulin mAb (Sigma-Aldrich), anti-GluA1 N-terminal antibody mAb (4.9D, made in-house), anti-GluA2 N-terminal antibody mAb (032.19.9, made in-house), anti-GluA2 phospho-S880 specific mAb (02.22.4, made in-house), anti-GluA2 phospho-Y876 specific mAb (045.10.5, made in-house), anti-PSD95 mAb (NeuroMab), anti-GluA3 pAb (JH4300, made in-house), anti-GRIP1 mAb (BD Biosciences), anti-GRIP1 pAb (Chemicon), and anti-GRIP1 pAb (JH2260, made in-house).

Techniques: Activity Assay, Expressing, Western Blot, MANN-WHITNEY

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: GRIP1 regulates synaptic plasticity and learning and memory

doi: 10.1073/pnas.2014827117

Figure Lengend Snippet: Grip1 KO mice exhibit impaired NMDAR-dependent LTP. (A) Representative Western blots of total cell lysates from hippocampus in Nestin-Grip1fl/fl mice (Grip1 KO) and control Grip1fl/fl littermates (WT). (B) Representative evoked EPSCs obtained from CA1 pyramidal neurons before and after LTP induction in response to 0.1-Hz stimulation of Schaffer collaterals. Dash lines represent baseline EPSCs. Solid lines represent EPSCs 40 min after LTP induction. (C) Averaged EPSC amplitudes normalized to baseline responses. Arrow indicates the pairing induction (200 pulses at 2 Hz paired with 0 mV depolarization). (D) Statistics of LTP at 30–50 min (n = 16 cells from 8 control Grip1fl/fl littermate group; n = 17 cells from 8 Nestin-Grip1fl/fl mice; Mann–Whitney test). (E) Averaged EPSC amplitudes at baseline and during induction. Data are presented as mean ± SEM *P < 0.05.

Article Snippet: The following antibodies were used: anti–beta-tubulin mAb (Sigma-Aldrich), anti-GluA1 N-terminal antibody mAb (4.9D, made in-house), anti-GluA2 N-terminal antibody mAb (032.19.9, made in-house), anti-GluA2 phospho-S880 specific mAb (02.22.4, made in-house), anti-GluA2 phospho-Y876 specific mAb (045.10.5, made in-house), anti-PSD95 mAb (NeuroMab), anti-GluA3 pAb (JH4300, made in-house), anti-GRIP1 mAb (BD Biosciences), anti-GRIP1 pAb (Chemicon), and anti-GRIP1 pAb (JH2260, made in-house).

Techniques: Western Blot, MANN-WHITNEY

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: GRIP1 regulates synaptic plasticity and learning and memory

doi: 10.1073/pnas.2014827117

Figure Lengend Snippet: Grip1 KO mice display impaired learning and memory. (A) Cartoon illustration of the IA task. (B) Quantifications of the latency to cross over to the dark chamber at training and 24 h later in CaMKII-Grip1fl/fl (Grip1 KO) and control Grip1fl/fl littermates (WT) (n = 15 WT; n = 13 Grip1 KO; Mann–Whitney U test). (C) Quantification of total, central, and peripheral ambulatory activities in open-field chambers (n = 15 WT; n = 13 Grip1 KO; Mann–Whitney U test). (D) Quantification of the anxiety index, calculated as the activity in the peripheral divided by the activity in the center for each mouse (n = 15 WT; n = 13 Grip1 KO; Student’s t test). Data are presented as mean ± SEM, n.s., not significant; ***P < 0.001.

Article Snippet: The following antibodies were used: anti–beta-tubulin mAb (Sigma-Aldrich), anti-GluA1 N-terminal antibody mAb (4.9D, made in-house), anti-GluA2 N-terminal antibody mAb (032.19.9, made in-house), anti-GluA2 phospho-S880 specific mAb (02.22.4, made in-house), anti-GluA2 phospho-Y876 specific mAb (045.10.5, made in-house), anti-PSD95 mAb (NeuroMab), anti-GluA3 pAb (JH4300, made in-house), anti-GRIP1 mAb (BD Biosciences), anti-GRIP1 pAb (Chemicon), and anti-GRIP1 pAb (JH2260, made in-house).

Techniques: MANN-WHITNEY, Activity Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: GRIP1 regulates synaptic plasticity and learning and memory

doi: 10.1073/pnas.2014827117

Figure Lengend Snippet: The GRIP1–GluA2 association is enhanced during LTP. (A) Representative Western blots of proteins from P2 in rat cortical neurons treated with (+) or without (−) glycine. (B) Quantification of phospho-Y876 and phospho-S880 levels following cLTP (n = 7 to 12; Student’s t test). (C) GluA2 was immunoprecipitated with specific GluA2 antibody from P2 from rat cortical neurons treated with (+) or without (−) glycine, followed by Western blot analysis of GRIP1 and GluA2. (D) Quantification of relative GRIP1–GluA2 interactions in P2 during cLTP (n = 10; Mann–Whitney U test). (E) Model of GRIP1 regulation of AMPAR trafficking during LTP. Data are presented as mean ± SEM. n.s., not significant; *P < 0.05; ***P < 0.001.

Article Snippet: The following antibodies were used: anti–beta-tubulin mAb (Sigma-Aldrich), anti-GluA1 N-terminal antibody mAb (4.9D, made in-house), anti-GluA2 N-terminal antibody mAb (032.19.9, made in-house), anti-GluA2 phospho-S880 specific mAb (02.22.4, made in-house), anti-GluA2 phospho-Y876 specific mAb (045.10.5, made in-house), anti-PSD95 mAb (NeuroMab), anti-GluA3 pAb (JH4300, made in-house), anti-GRIP1 mAb (BD Biosciences), anti-GRIP1 pAb (Chemicon), and anti-GRIP1 pAb (JH2260, made in-house).

Techniques: Western Blot, Immunoprecipitation, MANN-WHITNEY

Journal: The Journal of Neuroscience

Article Title: PICK1 Mediates Transient Synaptic Expression of GluA2-Lacking AMPA Receptors during Glycine-Induced AMPA Receptor Trafficking

doi: 10.1523/JNEUROSCI.5068-11.2012

Figure Lengend Snippet: Transient increase in PICK1 localization at endosomal compartments after glycine stimulation. A, Colocalization between GluA2 and PICK1 is transiently increased at endosomal compartments after stimulation with glycine. Dissociated hippocampal neurons were preloaded with Alexa Fluor–transferrin to label endosomes (red channel). Neurons were exposed to elevated glycine for 3 min, returned to normal medium for the specified time, fixed at 0, 5, 10, or 20 min after stimulus, and stained using anti-GluA2 (green channel) and anti-PICK1 (blue channel). Controls did not receive the stimulus. Representative images are shown for all conditions. Graph shows Manders' colocalization coefficients for the fraction of GluA2 colocalized with PICK1 within the transferrin compartment normalized to control. *p < 0.0125, ***p < 0.001. n = 3 independent experiments, with 10–15 cells in each. Scale bar, 5 μm. B, Colocalization between PICK1 and EEA1 is transiently enhanced in dendrites at 5 min after glycine stimulation. Dissociated hippocampal neurons were glycine stimulated as in A and stained using anti-EEA1 (green channel) and anti-PICK1 (red channel). Controls did not receive the stimulus. Representative images are shown for all conditions. Graph shows Manders' colocalization coefficients for the fraction of PICK1 colocalized with EEA1 normalized to control. **p < 0.01. n = 4 independent experiments, with 10–12 cells in each. Scale bar, 5 μm. C, Colocalization between PICK1 and the recycling endosomal marker Rab11 is transiently enhanced in dendrites at 5 min after glycine stimulation. Dissociated hippocampal neurons were glycine stimulated as in A and stained using anti-Rab11 (green channel) and anti-PICK1 (red channel). Controls did not receive the stimulus. Representative images are shown for all conditions. Graph shows Manders' colocalization coefficients for the fraction of PICK1 colocalized with Rab11 normalized to control condition. **p < 0.01, ***p < 0.001. n = 4 independent experiments, with 10–12 cells in each. Scale bar, 5 μm. D, Colocalization between PICK1 and the synaptic marker PSD95 is unchanged at 5 min after glycine stimulation but increases at 20 min. Dissociated hippocampal neurons were glycine stimulated as in A and returned to normal medium for the specified time, fixed at 5 or 20 min after stimulus, and stained using anti-PSD95 (green channel) and anti-PICK1 (red channel). Controls did not receive the stimulus. Representative images are shown for all conditions. Graph shows Manders' colocalization coefficients for the fraction of PICK1 colocalized with PSD95 normalized to control condition. *p < 0.025. n = 3 independent experiments, with 10–12 cells in each. Scale bar, 5 μm. E, Association between PICK1 and Rab11 analyzed by live imaging. Dissociated hippocampal neurons expressing GFP–Rab11 and mCherry–PICK1 were exposed to elevated glycine for 3 min, followed by washout with normal medium. Images were collected every minute, and fluorescence intensity for mCherry–PICK1 was measured within a mask generated from the GFP–Rab11 signal. Images show representative images from selected time points, and graph shows fluorescence normalized to baseline (before glycine application). n = 7. Scale bar, 2 μm.

Article Snippet: The following antibodies were used: N-terminal anti-GluA1, anti-GluA2, and anti-GluA3 (Millipore); anti-PICK1 (NeuroMab); anti-GRIP1 and anti-Rab11 (BD Biosciences); and anti-early endosomal antigen 1 (EEA1), anti-PSD95, and anti-tubulin (all from Sigma).

Techniques: Staining, Marker, Imaging, Expressing, Fluorescence, Generated

Journal: The Journal of Neuroscience

Article Title: PICK1 Mediates Transient Synaptic Expression of GluA2-Lacking AMPA Receptors during Glycine-Induced AMPA Receptor Trafficking

doi: 10.1523/JNEUROSCI.5068-11.2012

Figure Lengend Snippet: Calcium-sensing function of PICK1 is required for the regulation of GluA2 trafficking to the plasma membrane after glycine stimulation. A, Schematic diagram showing PICK1 mutants used. B, Calcium-sensing regions of PICK1 are involved in GluA2 trafficking to the plasma membrane after glycine stimulation. Dissociated hippocampal neurons expressing DD4,6AA–PICK1flag–IRES–EGFP or Δ380–390–PICK1flag–IRES–EGFP were stimulated with glycine for 3 min, returned to normal medium for the specified time, and fixed 5 or 20 min after stimulus. Controls did not receive the stimulus. Cells were processed for immunocytochemistry as described for Figure 1. *p < 0.025; n = 3 independent experiments, with 12–15 cells in each. Scale bar, 20 μm. C, Colocalization between WT–PICK1flag and the recycling endosomal marker Rab11 is transiently enhanced in dendrites at 5 min after glycine stimulation. Dissociated hippocampal neurons were glycine stimulated as in B and stained using anti-Rab11 (red channel) and anti-flag (green channel). Controls did not receive the stimulus. Representative images are shown for all conditions. Graphs show Manders' colocalization coefficients for the fraction of WT–PICK1flag colocalized with Rab11 normalized to control condition. *p < 0.05, n = 3 independent experiments, with 10–12 cells in each. Scale bar, 5 μm. D, Colocalization between DD4,6AA–PICK1flag and the recycling endosomal marker Rab11 is unaffected by glycine stimulation. Dissociated hippocampal neurons were glycine stimulated as in B and stained using anti-Rab11 (red channel) and anti-flag (green channel). Controls did not receive the stimulus. Representative images are shown for all conditions. Graphs show Manders' colocalization coefficients for the fraction of DD4,6AA–PICK1flag colocalized with Rab11 normalized to control condition. n = 3 independent experiments, with 10–12 cells in each. Scale bar, 5 μm. E, Colocalization between Δ380–390–PICK1flag and the recycling endosomal marker Rab11 is unaffected by glycine stimulation. Dissociated hippocampal neurons were glycine stimulated as in B and stained using anti-Rab11 (red channel) and anti-flag (green channel). Controls did not receive the stimulus. Representative images are shown for all conditions. Graphs show Manders' colocalization coefficients for the fraction of Δ380–390–PICK1flag colocalized with Rab11 normalized to control condition. n = 3 independent experiments, with 10–12 cells in each. Scale bar, 5 μm.

Article Snippet: The following antibodies were used: N-terminal anti-GluA1, anti-GluA2, and anti-GluA3 (Millipore); anti-PICK1 (NeuroMab); anti-GRIP1 and anti-Rab11 (BD Biosciences); and anti-early endosomal antigen 1 (EEA1), anti-PSD95, and anti-tubulin (all from Sigma).

Techniques: Expressing, Immunocytochemistry, Marker, Staining