Journal: Neuron

Article Title: Regulation of Thalamic and Cortical Network Synchrony by Scn8a

doi: 10.1016/j.neuron.2017.01.031

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Monoclonal mouse anti-GluR2 , Neuromab , Cat# 75-002; RRID: AB_2232661.

Techniques: Plasmid Preparation, Virus, Recombinant, Avidin-Biotin Assay, Software, Imaging

Journal: Scientific Reports

Article Title: PDI augments kainic acid-induced seizure activity and neuronal death by inhibiting PP2A-GluA2-PICK1-mediated AMPA receptor internalization in the mouse hippocampus

doi: 10.1038/s41598-023-41014-7

Figure Lengend Snippet: Effects of PDI knockdown on PP2A bindings to PDI and GluA2 in the hippocampus following KA injection. KA increases PDI:PP2A and GluA2:PP2A bindings in control siRNA-infused animals, but not in PDI siRNA-infused animals. ( a ) Representative Western blot images for the PDI:PP2A and GluA2:PP2A bindings. ( b – c ) Quantitative analyses of the effects of PDI siRNA on PP2A level ( b ) and PDI:PP2A binding ( c ) following KA injection (*, # p < 0.05 vs. control siRNA vs. saline; n = 7, respectively; Kruskal–Wallis test followed by Tukey post-hoc test).

Article Snippet: p-GRIA2 Y869/873/876 , Rabbit , Cell signaling (#3921) , 1:1,000 (WB).

Techniques: Knockdown, Injection, Control, Western Blot, Binding Assay, Saline

Journal: Scientific Reports

Article Title: PDI augments kainic acid-induced seizure activity and neuronal death by inhibiting PP2A-GluA2-PICK1-mediated AMPA receptor internalization in the mouse hippocampus

doi: 10.1038/s41598-023-41014-7

Figure Lengend Snippet: Effects of PDI knockdown on PICK1 bindings to GluA2 in the hippocampus following KA injection. PDI siRNA does not influence PICK1 level in both saline- and KA-treated groups. As compared to control siRNA, PDI siRNA increases GluA2A:PICK1 binding in saline-treated group. Although KA does not affect GluA2:PICK1 binding in control siRNA-infused group, it increases it in PDI siRNA-infused group. ( a ) Representative Western blot images for the GluA2:PICK1 binding. ( b – c ) Quantitative analyses of the effects of PDI siRNA on PICK1 level ( b ) and GluA2:PICK1 binding ( c ) following KA injection (*, # p < 0.05 vs. control siRNA vs. saline; n = 7, respectively; Kruskal–Wallis test followed by Tukey post-hoc test). ( d ) Scheme of the role of PDI in AMPAR internalization. PDI may reduce lead to reduction-induced PP2A activation, which would abolish PICK1-mediated AMPAR internalization by dephosphorylating GluA2 S880 and CaMKII T286 sites.

Article Snippet: p-GRIA2 Y869/873/876 , Rabbit , Cell signaling (#3921) , 1:1,000 (WB).

Techniques: Knockdown, Injection, Saline, Control, Binding Assay, Western Blot, Activation Assay

Journal: Scientific Reports

Article Title: PDI augments kainic acid-induced seizure activity and neuronal death by inhibiting PP2A-GluA2-PICK1-mediated AMPA receptor internalization in the mouse hippocampus

doi: 10.1038/s41598-023-41014-7

Figure Lengend Snippet: Primary antibodies used in the present study.

Article Snippet: p-GRIA2 Y869/873/876 , Rabbit , Cell signaling (#3921) , 1:1,000 (WB).

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons

doi: 10.3389/fcell.2021.708715

Figure Lengend Snippet: Characterization of differentiated PC12 cells and validation of single-molecule labeling. (A,B) Left, Intensity profiles of a single ATTO 488-labeled GluR2-AMPAR (A) and mGluR1 (B) signal. The arrows indicate single-step photobleaching. Right, Histogram showing the intensity value of every spot found in a recording of ATTO 488-labeled GluR2-AMPAR (A) and mGluR1 (B) , superimposed with a single fitted lognormal curve (blue line). (C) Representative trajectories of AMPAR molecules on somas and neurites. Scale bar = 2 μm. (D) The mean square displacement functions and trajectories represent AMPAR molecules with Brownian motion (red) and confined motion (blue). Scale bar = 0.1 μm. (E,F) The cumulative probability functions of D values of AMPAR (E) and mGluR1 (F) on neurites and somas ( n = 510–676 trajectories). *** p < 0.001.

Article Snippet: The specificity of the antibodies was also tested with control peptides (GluA2 179–193 peptide and mGluR1 501–516 peptide, Alomone Labs), and no immunoreactivity was observed ( ).

Techniques: Labeling

Journal: Frontiers in Cell and Developmental Biology

Article Title: Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons

doi: 10.3389/fcell.2021.708715

Figure Lengend Snippet: Effect of E2 on the surface movement of GluR2-AMPAR and mGluR1. (A) Effect of different concentrations of E2 on the diffusion coefficient (D, μm 2 /s) of GluR2-AMPAR (A) and mGluR1 (B) (% of vehicle treatment as the mean ± SEM, n = 425–1145 trajectories per group). (C,D) Line graphs depict changes in D of GluR2-AMPAR (C) and mGluR1 (D) molecules at different time points after the administration of the most effective concentration of E2 (% of vehicle treatment as the mean D ± SEM, n = 117–187 trajectories per time point). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The specificity of the antibodies was also tested with control peptides (GluA2 179–193 peptide and mGluR1 501–516 peptide, Alomone Labs), and no immunoreactivity was observed ( ).

Techniques: Diffusion-based Assay, Concentration Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons

doi: 10.3389/fcell.2021.708715

Figure Lengend Snippet: Effect of estrogen receptor modulation on the surface movement of GluR2-AMPAR. (A) Representative PCR gel electrophoresis image depicting the expression of estrogen receptor beta (ERβ) and G protein-coupled estrogen receptor 1 (GPER1) mRNA in dPC12. Estrogen receptor alpha (ERα) mRNA was not detected. (B) Histograms demonstrate the mean D AMPAR as a percentage of vehicle control on somas and neurites in the presence of the estrogen receptor, β (ERβ) agonist diarylpropionitrile (DPN), a GPER1 agonist (G1), G1+DPN together, a GPER1 antagonist (G15) and G15+E2 (with 100 pM of E2 on the somas and 100 nM of E2 on the neurites) (mean ± SEM; n = 215–641 trajectories). *** p < 0.001.

Article Snippet: The specificity of the antibodies was also tested with control peptides (GluA2 179–193 peptide and mGluR1 501–516 peptide, Alomone Labs), and no immunoreactivity was observed ( ).

Techniques: Nucleic Acid Electrophoresis, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons

doi: 10.3389/fcell.2021.708715

Figure Lengend Snippet: The GluR2-AMPAR/GPER1 ratio and molecular distance between GPER1 and GluR2-AMPAR in the membrane. (A) STORM images depicting immunolabeled AMPAR (magenta) and GPER1 (cyan) molecules on dPC12. Dashed lines delineate the borders of the neurites and somas. Scale bar = 2 μm; inset Scale bar = 0.5 μm. (B) The ratio between the number of GPER1 and AMPAR molecules (GPER1/GluR2-AMPAR) on the neurites and somas ( n = 11 somas or neurites). (C1) Photomicrographs depict GPER1 immunoreactivity (visualized with STED microscopy) in dPC12 after 10 min of vehicle (left) or of 100 nM of E2 treatment (right). Scale bar = 2 μm. (C2) One 2 μm 2 (between parallel white bars) and one 10 μm 2 (to the left) areas were selected within each ROI for the membrane and cytoplasmic regions of each cell, respectively. Integrated density was calculated and normalized to the area. Scale bar = 0.5 μm. (D) Dual labeling of plasma membrane and GPER1 molecules defines the membrane regions (approximately 1 μm wide). Scale bar = 0.5 μm. (E) Line graph of the fluorescent intensity calculated from the magnified STED inserts (C2). (F) Integrated density graphs of GPER1 show the effect of vehicle and 100 nM of E2 treatment in the membrane and in the cytoplasm ( n = 15 cells were evaluated in each group). * p < 0.05.

Article Snippet: The specificity of the antibodies was also tested with control peptides (GluA2 179–193 peptide and mGluR1 501–516 peptide, Alomone Labs), and no immunoreactivity was observed ( ).

Techniques: Immunolabeling, Microscopy, Labeling

Journal: Frontiers in Cell and Developmental Biology

Article Title: Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons

doi: 10.3389/fcell.2021.708715

Figure Lengend Snippet: Effect of E2 on the surface movement of GluR2-AMPA on primary hippocampal neurons. (A) Photomicrograph shows a primary hippocampal neuron labeled with homer-1 (synapse) and β-III tubulin (neuron). Scale bar = 10 μm, insert Scale bar = 2 μm. (B) Dual color STED image of a hippocampal neuron overlayed to differential interference contrast microscopy image depicts live-cell synapse labeling MitoTracker Deep Red (red) and presynaptic protein bassoon (green). Scale bar = 1 μm. (C) Distribution of D values of extrasynaptic and synaptic GluR2-AMPAR under control conditions (median ± IQR, n = 754 extrasynaptic trajectories and n = 104 synaptic trajectories). (D) Effect of E2 (100 pM and 100 nM) on D of extrasynaptic and synaptic GluR2-AMPA with or without chemical LTP (cLTP) induced by glycine/picrotoxin (gly/pic) (% of vehicle treatment as the mean ± SEM; n = 742–928 extrasynaptic trajectories and n = 104–155 synaptic trajectories). (E,F) Effect of vehicle, E2 (100 n, 100 pM) with or without cLTP (gly/pic) on synaptic dwell time (mean ± SEM (s); n = 104–155) (E) and relative surface distribution of synaptic GluR2-AMPAR content (synaptic/total GluR2-AMPA molecule trajectories) (mean ± SEM, n = 8–18 recordings) (F) . * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The specificity of the antibodies was also tested with control peptides (GluA2 179–193 peptide and mGluR1 501–516 peptide, Alomone Labs), and no immunoreactivity was observed ( ).

Techniques: Labeling, Microscopy

Journal: Autophagy

Article Title: Table 1.

doi: 10.1080/15548627.2025.2597463

Figure Lengend Snippet: Autophagy-deficient PVALB-interneurons exhibit an altered synaptic composition, resulting in functional adaptations. (A) Representative images of surface GRIA2/GluA2 immunoreactivity (green) in hippocampal CA1 PVALB cells (magenta) from control ( Atg5 f/f ) vs. Pvalb-atg5 mice. Scale bar: 10 µm. (B) Quantification of the mean surface GRIA2/GluA2 intensity in control versus Pvalb-atg5 PVALB cells in hippocampal CA1. Results are expressed as a percentage of control values. Statistical analysis was performed using an unpaired, two-tailed Student’s t test (** p = 0.0028, t = 3.737, df = 12). Bars represent mean values ±SEM. N = 7 mice per genotype. (C) Representative images of total GRIA2/GluA2 immunoreactivity (green) in hippocampal CA1 PVALB cells (magenta) from control ( Atg5 f/f ) vs. Pvalb-atg5 mice. Scale bar: 10 µm. (D) Quantification of the mean total GRIA2/GluA2 intensity in control versus Pvalb-atg5 PVALB cells in hippocampal CA1. Results are expressed as a percentage of control values. Statistical analysis was performed using an unpaired, two-tailed Student’s t test ( ns p = 0.4154, t = 0.9076, df = 4). Bars represent mean values ±SEM. N = 3 mice per genotype. (E-I) Patch clamp recordings of AMPA (E) and NMDA (F) currents from CA1 PVALB cells from control ( Pvalb-TdT ) and Atg5 -deficient PVALB interneurons ( Pvalb-atg5-TdT ). These data were also plotted as the AMPA:NMDA ratio for controls and Pvalb-atg5-TdT animals, where all values were normalized to the control (G). (H) represents the average AMPA (black) and NMDA (gray) traces recorded at + 40 mV for control and Pvalb-atg5-TdT cells. (I) Reduced AMPA currents over NMDA currents in Pvalb-atg5-TdT mice when compared to controls. Black line indicates R 2 = 1. Statistical analyses were performed using a Student’s t test (for AMPA currents: * p = 0.0464, t = 2.100, df = 24; for NMDA currents, ns p = 0.3955, t = 0.8651, df = 24; for AMPA:NMDA ratios: * p = 0.0348, t = 2.238, df = 24). Bars represent mean values ± SEM. N = 3 animals per genotype ( Pvalb-TdT, n = 14; Pvalb-atg5-TdT , n = 16). (J) Current-clamp sample traces (400 pA injection, top) and the action potentials evoked by current steps (bottom) from PVALB neurons, from slices of control (Pvalb-TdT , RMP = −63 mV) and mutant Pvalb-atg5-TdT (RMP = −62 mV) mice. (K) Input-output plots from control (Pvalb-TdT) ( n = 14, N = 3) and Pvalb-atg5-TdT cells ( n = 16, N = 3). Statistical analysis was performed using a two-way repeated measures ANOVA followed by Sidak post hoc test; F(5,140) = 23.85 (**** p < 0.005). Bars represent mean values ± SEM. (L) Comparison of capacitance, resting membrane potential, action potential threshold and amplitude in control (Pvalb-TdT) ( n = 14, N = 3) and Pvalb-atg5-TdT cells ( n = 16, N = 3). Statistical analyses were performed using a Student’s t test (ap threshold, * p = 0.023, t = 2.398, df = 28). Bars represent mean values ± SEM. (M) Representative traces of sIPSC from slices of control (Pvalb-TdT) and mutant Pvalb-atg5-TdT mice recorded at + 5 mV. (N-O) Bar graphs and cumulative plots related tothe frequency and amplitude of sIpscs from control (Pvalb-TdT) ( n = 14, N = 3) and mutant Pvalb-atg5-TdT mice ( n = 16, N = 3). Statistical analysis was performed using an unpaired, two-tailed Student’s t test (* p = 0.0298, t = 2.289, df = 28 and ns p = 0.8654, t = 0.1710, df = 28, respectively). Bars represent mean values ± SEM. (P) Representative traces of paired-pulse ratio (IPSC 2 :IPSC 1 ) from three-month old control (Pvalb-TdT) and mutant Pvalb-atg5-TdT mice recorded at + 5 mV. (Q) Graph showing the paired-pulse ratio (IPSC 2 :IPSC 1 ) from control (Pvalb-TdT) ( n = 8, N = 2) and mutant Pvalb-atg5-TdT mice ( n = 12, N = 2). Statistical analysis was performed using an unpaired, two-tailed Student’s t test (* p = 0.0310, t = 2.340, df = 18). Bars represent mean values ± SEM. (R) Representative traces of excitation to inhibition ratio (EPSC −60 mV :IPSC +5 mV ) from control (Pvalb-TdT) and mutant Pvalb-atg5-TdT mice. (S) Graph showing the excitation to inhibition ratio (EPSC:IPSC) from control (Pvalb-TdT) ( n = 8, N = 2) and mutant Pvalb-atg5-TdT ( n = 14, N = 3). Statistical analysis was performed using an unpaired, two-tailed Student’s t test (* p = 0.0351, t = 2.261, df = 20). Bars represent mean values ± SEM.

Article Snippet: GRIA2/GluA2 (extracellular) , Guinea pig , Alomone labs , AGC-005-GP , 1:500 (IHC).

Techniques: Functional Assay, Control, Two Tailed Test, Patch Clamp, Injection, Mutagenesis, Comparison, Membrane, Inhibition

Journal: BMC Biology

Article Title: CLARITY increases sensitivity and specificity of fluorescence immunostaining in long-term archived human brain tissue

doi: 10.1186/s12915-023-01582-6

Figure Lengend Snippet: Detailed information on the 30 primary antibodies used

Article Snippet: G 7 , Postsynapse (excitatory) , GLUA2 (GRIA2) , ms , 1:200 , SySy Cat# 182 111, RRID:AB_10645888 , .

Techniques: Sequencing, Tandem Mass Spectroscopy

Journal: BMC Biology

Article Title: CLARITY increases sensitivity and specificity of fluorescence immunostaining in long-term archived human brain tissue

doi: 10.1186/s12915-023-01582-6

Figure Lengend Snippet: Increased sensitivity of antibodies applied on CLARITY-processed sections. a Cleared (orange) and non-cleared (blue) frontal cortex sections from case p1 were stained for CAMKIIA and imaged 15 µm deep in 1.8 µm steps using a confocal microscope. Z-level I (z = 0 µm), III (z = 3.6 µm), and VI (z = 9 µm) are provided as single images (left). Mean intensities of each z-step were exported with LAS-X software from two stacks (cortex layer V), and the mean was plotted in dependency of the z-level (right). A side view (xz scan) is shown in the middle. Scale bars, 10 µm. b CAMKIIA-positive surfaces in the neuropil outside dendrites of cases p1 (perfusion; filled circle) and p2 (white circle) were counted in z-levels I (z = 0 µm), III (z = 3.6 µm), and VI (z = 9 µm) of two z-stacks per case and condition (acquired with identical settings). In total, two regions of interest (ROIs) were analyzed per image in Imaris with constant parameters for plus and minus CLARITY per z-plane. The mean ratios between plus and minus CLARITY (y axis) of two technical replicates are plotted; a ratio of 1 (black line) indicates identical surface numbers for plus and minus CLARITY. c Mean intensity of GLUA2 staining on case p1 was plotted in dependency of the z-level. Acquisition strategy is the same as outlined in panel (a). Scale bars, 5 µm. d More sensitive detection of GLUA2 puncta after CLARITY, analysis as described in panel (b)

Article Snippet: G 7 , Postsynapse (excitatory) , GLUA2 (GRIA2) , ms , 1:200 , SySy Cat# 182 111, RRID:AB_10645888 , .

Techniques: Staining, Microscopy, Software

Journal: BMC Biology

Article Title: CLARITY increases sensitivity and specificity of fluorescence immunostaining in long-term archived human brain tissue

doi: 10.1186/s12915-023-01582-6

Figure Lengend Snippet: Super-resolution microscopy with STED and dSTORM imaging of synaptic and neuronal markers on human brain tissue after CLARITY. a Synaptic GLUA2 in the cerebral cortex acquired in confocal (upper left) and STED mode (lower right). Scale bar, 1 µm. b Insets (boxed area from (a)) provided in higher magnification unveil postsynaptic membrane morphologies like disk shape as seen in a side view on a synapse (asterisks, left column) and the detailed localization of GLUA2 within a single synaptic spot as seen in a top view on a synapse (right column). A line intensity profile (right part of (b)) across the spot surface demonstrates the intensity fluctuation along the spot (white line in the boxed areas of (b)). Mean intensities (gray values) were normalized on the highest value. a.u. = arbitrary unit. Scale bar, 1 µm. c MAP2 staining in a cerebral cortex section. STED and confocal modes of the same neuron are shown in comparison as in panel (a). Scale bar, 1 µm. d Insets (boxed area from (c)) provided in higher magnification show structural details in the STED mode. The intensity profile across the white lines in the images is plotted on the right. Scale bar, 0.5 µm. Sections for MAP2 and GLUA2 staining were derived from the superior frontal gyrus of perfusion-fixed cases. In (a)-(d), pixel size for confocal images is 50 nm. e Schematic drawing showing expected precision imaging of synaptic compartments in super-resolution images (upper left). SYP/GLUA1 co-staining in STED microscopy. The channels are shown individually (left and middle) and as overlay (right). Here, sections were incubated in hydrogel solution for two days, total clearing time was three days. Scale bars, 200 nm. f SYP/GLUA1 co-staining in dSTORM microscopy (left). An intensity profile across a synapse (rectangle in the left image, width of ten pixels) is shown on the right. Sections were incubated in hydrogel solution for one day, total clearing time was two days. Scale bar, 200 nm. All sections in (e)-(f) were derived from perfusion-fixed cases

Article Snippet: G 7 , Postsynapse (excitatory) , GLUA2 (GRIA2) , ms , 1:200 , SySy Cat# 182 111, RRID:AB_10645888 , .

Techniques: Super-Resolution Microscopy, Imaging, Membrane, Staining, Comparison, Derivative Assay, Microscopy, Incubation

Journal: Nature

Article Title: Electrical and synaptic integration of glioma into neural circuits.

doi: 10.1038/s41586-019-1563-y

Figure Lengend Snippet: Fig. 4 | Glioma membrane depolarization promotes glioma progression. a, Optogenetic model for glioma depolarization. Blue dots represent ChR2-expressing glioma cells; light blue rectangle denotes region of analysis. P, postnatal day. b, Proliferation index of SU-DIPG- XIII-FL-ChR2 xenografts after mock stimulation or blue light stimulation, measured as percentage of GFP+/HNA+ cells expressing Ki67 (mock stim, n = 8; stim, n = 9 mice). c, As in b but for SU-DIPG-VI-ChR2 xenografts (n = 6 mice per group). d, Representative confocal micrographs from c, illustrating proliferation of SU-DIPG-VI-ChR2 xenografts. Red denotes human nuclei staining by HNA; white denotes Ki67. Scale bar, 50 µm. e, f, Kaplan–Meier survival curves of SU-DIPG-XIII-P* (P denotes pontine tumour) xenografts that overexpress GFP-only (green) or GluA2- WT-GFP (red) (e) and GFP-only (in 80% of cells, green) or GluA2-DN- GFP (in 80% of cells, blue) (f) (n = 5 mice per group). g, Competitive outgrowth of non-GluA2-DN-GFP-expressing cells in f, determined by

Article Snippet: We introduced SpeI (5′-TAAGCAACTAG TATGCAAAAGATTATGCAT-3′) and XmaI (5′-TGCTTACCCGGGC TAAATTTTAACACTTTCGAT-3′) restriction sites in full-length human GRIA2 clone (GluA2 WT) obtained from Origene (RC212599) by PCR.

Techniques: Membrane, Expressing, Staining