Journal: bioRxiv

Article Title: Expansion microscopy at one nanometer resolution

doi: 10.1101/2022.08.03.502284

Figure Lengend Snippet: a-c , Synaptic vesicles were labeled live using an antibody against a luminal epitope of synaptotagmin 1 (Syt1, magenta). The vesicular glutamate transporter (vGluT1, blue) and PSD95 (gray) were immunostained using an antibody and a nanobody, respectively. a , Recently endocytosed vesicle exhibiting circular morphology. b , Readily retrievable pool molecules form patches containing Syt1/vGluT1 (top), which are dispersed by cholesterol extraction using MβCD (bottom). c , MβCD causes molecules to spread across larger areas (left: N = 22-19, 2 independent experiments, p < 0.0044, Mann-Whitney test; right: N = 22-22, 2 independent experiments, p = 0.8937), although the signal per vesicle (the Syt1 copy number) remains unchanged. d , A visualization of PSDs (top and side views), after immunostaining PSD95 with the same nanobody used in a-c, and Shank2 and Homer1 with specific antibodies. The graph indicates the axial positioning, which agrees well with the literature . N = 11 measurements for each protein, 2 independent experiments; symbols show the medians, SEM and SD. e , Side view of a postsynapse displaying PSD95, MAP2 and two glutamate receptors (GluR2, AMPA type, and GluN2b, NMDA type). f , ONE images of PSD95 (top views), before or after the addition of 10% 1,6-hexanediol (Hex). g , Line scans through the PSD95 stainings shown in panel f. h , An analysis of PSD95 spot profiles; N = 10-7 synapses, Friedman test followed by Dunn-Sidak testing, p = 0.0027; the error bars show the SEM. For details on the analysis, see .

Article Snippet: The primary antibodies used were anti synaptotagmin1 (SYT1, #105011 Synaptic Systems), anti Homer1 (#160 003, Synpatic Systems), anti Shank2 (#162204 Synaptic Systems), anti GluR2 (Alomone Labs, #AGC-005, Jerusalem, Israel), anti GluN2b (Neuromab 75-101, California, USA), anti MAP2 (Novus Biologicals #NB300-213), anti vGluT1 (#135304, Synaptic Systems), anti Bassoon (#ADI-VAM-PS003-F, Enzo, New York, USA).

Techniques: Labeling, MANN-WHITNEY, Immunostaining

Journal: Annals of Translational Medicine

Article Title: The recycling of AMPA receptors/GABAa receptors is related to neuronal excitation/inhibition imbalance and may be regulated by KIF5A

doi: 10.21037/atm-22-4337

Figure Lengend Snippet: Expression of KIF5A, GluR2 and beta 2+3 subunits of gamma aminobutyric acid receptors (Gabrb2+3) in an in vivo model of seizures. (A) EEG results: There was no epileptic discharge in the Ctl group after the injection of saline; however, epileptic discharge was observed in the Sez group after the injection of PTZ. (B) Total protein expression: the gray level of the total protein expression bands was normalized with GAPDH, and the total protein expression levels of KIF5A, GluR2 and Gabrb2+3 in the hippocampus of the Sez group did not change significantly (n=6 in each group, vs. Ctl, P>0.05). (C) Surface protein expression: The gray level of the total protein expression bands was normalized with Sodium/potassium-transporting ATPase subunit alpha-1 (ATP1A1). In the Sez group, the expression of GluR2 on the surface increased significantly to 181.74%±14.44% ( vs. Ctl, # , P<0.01). Conversely, the expression of GluR2 on the surface decreased to 19.62%±8.01% ( vs. Ctl, # , P<0.01). KIF5A, kinesin superfamily proteins 5A; GluR2, glutamate receptors subunit-2; Ctl, control; Sez, seizures; PTZ, pentylenetetrazol.

Article Snippet: The primary antibodies used include anti-KIF5A antibody 1:1,000 (SANTA, sc-376452), anti-GluR2 antibody 1:1,000 (BOSTER, PB9205), and anti-GABAaR β2+3 antibody 1:500 (Bioss, bs-12066R).

Techniques: Expressing, In Vivo, Injection

Journal: Annals of Translational Medicine

Article Title: The recycling of AMPA receptors/GABAa receptors is related to neuronal excitation/inhibition imbalance and may be regulated by KIF5A

doi: 10.21037/atm-22-4337

Figure Lengend Snippet: Receptor recycling assay (IF). The recycling ratio of GluR2 was 0.30±0.05 in the Ctl group and 0.60±0.07 in the Mg 2+ -free group (7 cells per group, vs. Ctl, # , P<0.01), and the recycling ratio of Gabrb2+3 was 0.49±0.04 in Ctl group and 0.32±0.05 in the Mg 2+ -free group (7 cells per group, vs. Ctl, # , P<0.01) (×600). Scale Bar: 50 µm. IF, immunofluorescence; Ctl, control.

Article Snippet: The primary antibodies used include anti-KIF5A antibody 1:1,000 (SANTA, sc-376452), anti-GluR2 antibody 1:1,000 (BOSTER, PB9205), and anti-GABAaR β2+3 antibody 1:500 (Bioss, bs-12066R).

Techniques: Immunofluorescence

Journal: Annals of Translational Medicine

Article Title: The recycling of AMPA receptors/GABAa receptors is related to neuronal excitation/inhibition imbalance and may be regulated by KIF5A

doi: 10.21037/atm-22-4337

Figure Lengend Snippet: Interaction between KIF5A and GluR2 and Gabrb2+3 in the seizure model. (A) Co-ip results: The protein bands showed the co-ip levels of KIF5A, GluR2, and Gabrb2+3, and normalized with the protein levels of KIF5A. The GluR2 level of KIF5A pull-down in the hippocampi of the rats in the Sez group increased to 130.42%±53.24% (n=6 per, vs. Ctl, *, P<0.05). However, the Gabrb2+3 level of KIF5A decreased to 50.86%±5.33% in the Sez group (n=6 per group, vs. Ctl, # , P<0.01). (B) IF results: the Pearson’s correlation coefficients (PCC) of KIF5A/GluR2 was 0.40±0.19 in the Ctl group and 0.87±0.11 in the Mg 2+ -free solution group (n=6 per group, vs. Ctl, # , P<0.01) (×400). Scale Bar: 100 µm. (C) IF results: the PCC of KIF5A/Gabrb2+3 was 0.97±0.02 in the Ctl group and 0.32±0.11 in the Mg 2+ -free solution group (n=6 per group, vs. Ctl, # , P<0.01) (×400). Scale Bar: 100 µm. IF, immunofluorescence.

Article Snippet: The primary antibodies used include anti-KIF5A antibody 1:1,000 (SANTA, sc-376452), anti-GluR2 antibody 1:1,000 (BOSTER, PB9205), and anti-GABAaR β2+3 antibody 1:500 (Bioss, bs-12066R).

Techniques: Co-Immunoprecipitation Assay, Immunofluorescence

Journal: Neuron

Article Title: Spatial representations of granule cells and mossy cells of the dentate gyrus

doi: 10.1016/j.neuron.2016.12.026

Figure Lengend Snippet: A–C) One mossy cell with 3 firing fields. A-B) Same convention used for Figure 5A–B. Scale bars in B: 1 sec (horizontal) and 1 mV (vertical). C) Top middle, the morphology and location of the recorded cell. The inset to the right shows a magnified view, where large spines (“thorny excrescences”) covering the soma and proximal dendrites, typical of mossy cells, can be clearly seen. The camera lucida reconstructed morphology of the mossy cell is shown at left. The boundaries of GCL and CA3 are represented with blue lines. Bottom, antibody staining of the cell (left to right: merge, Neurobiotin, GluR2/3, DAPI). The labeled cell (indicated with arrows) is positive for GluR2/3. Scale bar: 50 µm. (D–F) A mossy cell with 2 firing fields. Note that only the soma and parts of the dendritic arbor of the cell were recovered after labeling. The identity of this mossy cell was confirmed by the location of the cell body (in the hilus), the GluR2/3+ signal, as well as some large spines on the cell body (F, top right panel). (G–I) A mossy cell close to the lower blade of the GCL with a single field. Although only one labeling attempt was made, three cells were labeled in this rat. We believe that the recorded cell was a mossy cell because the deepest labeled cell was adjacent to the pipette tip, was located in the hilus (I, top), and was GluR2/3+ (I, bottom). The 6 ms burst indices for these 3 mossy cells were 0.17 (B), 0.11 (E) and 0.19 (H), respectively.

Article Snippet: If one hilar cell was labelled, the slices were further processed for GluR2/3 immunoreactivity (anti-GluR2/3 antibody, Cat. #AB1506, Millipore; Alexa Fluor® 546 Goat Anti-Rabbit IgG (H+L), Cat. #A-11010, ThermoFisher Scientific) to determine whether it was a mossy cell or an interneuron.

Techniques: Staining, Labeling, Transferring

Journal: Neuron

Article Title: Spatial representations of granule cells and mossy cells of the dentate gyrus

doi: 10.1016/j.neuron.2016.12.026

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: If one hilar cell was labelled, the slices were further processed for GluR2/3 immunoreactivity (anti-GluR2/3 antibody, Cat. #AB1506, Millipore; Alexa Fluor® 546 Goat Anti-Rabbit IgG (H+L), Cat. #A-11010, ThermoFisher Scientific) to determine whether it was a mossy cell or an interneuron.

Techniques: Recombinant, Plasmid Preparation, Software

Journal: Nature Communications

Article Title: Prefrontal cortex molecular clock modulates development of depression-like phenotype and rapid antidepressant response in mice

doi: 10.1038/s41467-024-51716-9

Figure Lengend Snippet: a , b Representative western blots and quantitative data of synaptic AMPA receptors subunits GluA1 and GluA2 levels normalized to Homer1b/c and PSD95 in mPFC of WT mice 24 h after SR10067 application ( a ), and 6 h and 24 h post SR1078 injection ( b ). ( n = 5, a : two-tailed Student’s t test: * P = 0.0422 for GluA1, ** P = 0.0069 for GluA2, b : one-way ANOVA with Bonferroni post hoc test: * P < 0.05, *** P < 0.001). c Experimental design: WT mice were subjected to the CDM protocol before undergoing implantation of ECoG, LFP and EMG recording electrodes. After recovery, mice were recorded in their home cage over 32 h from ZT0, with treatment (vehicle/ketamine/SR1078) administered at ZT06. d , e Delta power (0.5–4 Hz) of LFP signal recorded from the mPFC ( d ) and ECoG signal ( e ) recorded during slow wave sleep (SWS) across 12 h:12 h LD conditions ( n = 14 mice CDM/vehicle; n = 6 SR1078; n = 4 KET; repeated measures mixed-effects model two-way ANOVA with Bonferroni post hoc test * P < 0.05, ** P < 0.01 vs CDM/vehicle). Black arrow indicates time of treatment administration. f Schema summarizing the effects of RORα/γ activation on BMAL1, Homer1a and synaptic AMPAR expression in the mPFC and the relation to depression-like behavior. Data are presented as mean ± SEM and the individual data points are depicted. See also Supplementary Fig. and Supplementary Data . Source data are provided as a Source Data file.

Article Snippet: The membranes were blocked with 5% non-fat dry milk in TBS-T (1% Tween20 in Tris-buffered saline (TBS)) and afterward incubated with the respective primary antibodies diluted in TBS (mouse anti-GluR1-NT, Merck (MAB2263), 1:1000; rabbit anti-GluR2, Merck (AB1768-I), 1:1000; rabbit anti-Homer1, Merck (ABN37), 1:1000; mouse anti-PSD95, Merck (MABN68), 1:1000; rabbit anti-mouse Per2, Alpha Diagnostics (PER21-A), 1:1000; rabbit anti-BMAL1, Thermo Fisher Scientific PA1-523, 1:1000; mouse anti-Histone H2b, Euromedex (IG-H2-2A8), 1:500 overnight at 4 °C.

Techniques: Western Blot, Injection, Two Tailed Test, Activation Assay, Expressing

Journal: EMBO Reports

Article Title: Hyperfunction of post-synaptic density protein 95 promotes seizure response in early-stage aβ pathology

doi: 10.1038/s44319-024-00090-0

Figure Lengend Snippet: ( A , B ) Immunocytochemistry and quantification showing dendritic marker MAP2, total (t) and surface (s) GluA1 ( A ) and GluA2 ( B ) from WT primary cortical neuron cultures treated with Aβ 1-42 (Aβ42, 1 µM) or scrambled peptide (Scr, 1 µM) for 2 h at DIV 12–14. For GluA1, * P = 0.0192 for surface level, P = 0.42 for total level, and n = 17 and 18 cells from two independent cultures treated with Scr and Aβ42, respectively. For GluA2, ** P = 0.0025 for surface level, P = 0.1584 for total level, and n = 16 and 20 cells from two independent cultures treated with Scr and Aβ42, respectively. ( C , D ) Immunocytochemistry and quantification showing dendritic marker MAP2, total (t) and surface (s) GluA1 ( C ) and GluA2 ( D ) from PSD-95 +/− primary cortical neuron cultures treated with Aβ 1-42 (Aβ42, 1 µM) or scrambled peptide (Scr, 1 µM) for 2 h at DIV 12–14. For GluA1, P = 0.4539 for surface level, P = 0.1311 for total level, and n = 14 and 13 cells from two independent cultures treated with Scr and Aβ42, respectively. For GluA2, P = 0.6643 for surface level, P = 0.659 for total level, and n = 13 and 14 cells from two independent cultures treated with Scr and Aβ42, respectively. Data Information: significance was determined by Student’s t test ( A , C ) or Mann–Whitney U test ( B , D ). Data are represented as mean ± SEM with * P < 0.05, ** P < 0.01, ns non-significant. Scale bar: 5 μm. .

Article Snippet: Rabbit anti-GluA2-C-terminus antibody (#SAB4501295) is from Sigma.

Techniques: Immunocytochemistry, Marker, MANN-WHITNEY