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: 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: bioRxiv

Article Title: Single-molecule analysis reveals the rapid effect of estradiol on the surface movement of AMPAR in live neurons

doi: 10.1101/602813

Figure Lengend Snippet: Characterization of differentiated PC12 cells. ( a ) Photomicrograph depicting PC12 cells after 4 days of NGF treatment and ( b ) immunofluorescence staining of microtubule-associated protein 2 (MAP2) and β-III tubulin. Scale bars: 20 µm. ( c, d ) Representative recording of a whole-cell current-clamp step protocol obtained from PC12 neurons. ( c ) Application of increasing current steps in PC12 neuronal culture resulted in action potential generation. ( d ) Trace representing membrane potential changes in PC12 neurons. ( e, f ) Intensity profiles of a single ATTO 488-labeled GluR2-AMPAR and mGluR1 signal. The arrows indicate single-step photobleaching. ( g, h ) Histogram showing the intensity value of every spot found in a recording superimposed with a single fitted Gaussian curve (blue line).

Article Snippet: Before single-molecule imaging, PC12 neurons were incubated in dRPMI with ATTO 488-labeled antibodies directed against the extracellular N-terminal domain of rat GluR2 (1:100, Alomone Labs) or against the extracellular N-terminal domain of rat mGluR1 (1:100, Alomone Labs) at 37°C for 6 minutes.

Techniques: Immunofluorescence, Staining, Membrane, Labeling, Patch Clamp

Journal: bioRxiv

Article Title: Single-molecule analysis reveals the rapid effect of estradiol on the surface movement of AMPAR in live neurons

doi: 10.1101/602813

Figure Lengend Snippet: Single-molecule imaging of ATTO 488-labeled GluR2-AMPAR and mGluR1 receptors on neurites and on somas of PC12 neurons. ( a ) Representative trajectories of AMPAR molecules on somas and neurites. Scale bar: 2 µm. ( b ) The mean square displacement functions and trajectories represent AMPAR molecules with Brownian motion (red) and confined motion (blue). Scale bars: 0.1 µm. The cumulative probability functions of D values of AMPAR ( c ) and mGluR1 ( d ) on neurites and on somas are shown (n= 330-500 trajectories). **p<0.01, ***p<0.001

Article Snippet: Before single-molecule imaging, PC12 neurons were incubated in dRPMI with ATTO 488-labeled antibodies directed against the extracellular N-terminal domain of rat GluR2 (1:100, Alomone Labs) or against the extracellular N-terminal domain of rat mGluR1 (1:100, Alomone Labs) at 37°C for 6 minutes.

Techniques: Imaging, Labeling

Journal: bioRxiv

Article Title: Single-molecule analysis reveals the rapid effect of estradiol on the surface movement of AMPAR in live neurons

doi: 10.1101/602813

Figure Lengend Snippet: Effect of E2 on the surface trafficking of GluR2-AMPAR and mGluR1. The line graphs depict changes in the diffusion coefficient (D) of GluR2-AMPAR ( a ) and mGluR1 ( b ) molecules after vehicle or 100 pM E2 administration at different time points (mean ± SEM, n=100-128 trajectories per time point). ( c ) Distributions of D (as the percentage of vehicle control) of GluR2-AMPAR (average of 20-minute measurement) in the medium (dRPMI) or after administration of E2 at different concentrations (as the percent of the vehicle treatment expressed as the median ± 25-75%, n= 426-484 trajectories per group). ( d ) Distributions of D values of AMPAR on neurites in the presence of the estrogen receptor β (ERβ) agonist diarylpropionitrile (DPN), a GPER1 agonist (G1) and a GPER1 antagonist (G15) (expressed as the median ± 25-75%, n=427-468 trajectories). ( e ) Stochastic optical reconstruction microscopy images depicting immunolabeled AMPAR (magenta) and GPER1 (cyan) molecules on PC12 neurons. The dashed lines delineate the border between the neurite and soma. ( f ) Distributions of the minimal distance (greater than 500 nm) between GluR2-AMPAR and GPER1 after vehicle or E2 treatment (expressed as the median ± 25-75%, n=203-657). Scale bar: 2 µm; insert scale bar: 0.5 µm. **p<0.01, ***p<0.001

Article Snippet: Before single-molecule imaging, PC12 neurons were incubated in dRPMI with ATTO 488-labeled antibodies directed against the extracellular N-terminal domain of rat GluR2 (1:100, Alomone Labs) or against the extracellular N-terminal domain of rat mGluR1 (1:100, Alomone Labs) at 37°C for 6 minutes.

Techniques: Diffusion-based Assay, Control, Microscopy, Immunolabeling, Immunofluorescence

Journal: bioRxiv

Article Title: Single-molecule analysis reveals the rapid effect of estradiol on the surface movement of AMPAR in live neurons

doi: 10.1101/602813

Figure Lengend Snippet: Effect of estrogen receptor modulation on surface trafficking of AMPAR. ( a ) Representative PCR gel electrophoresis image depicting the expression of estrogen receptor mRNA (with a lack of expression of estrogen receptor alpha (ERα), estrogen receptor beta (ERβ), and G protein-coupled estrogen receptor 1 (GPER1)) in PC12 neurons. ( b ) Effect of E2 on the D value of mGluR1 (as the percent of vehicle treatment expressed as the median ± 25-75%, n= 840-860 trajectories per group). ( c ) Table showing the effect of ERβ agonist (DPN), GPER1 agonist (G1) and GPER1 antagonist (G15) treatment on D (µm 2 /s) values of GluR2-AMPAR molecules (expressed as the median ± 25-75% (in brackets), n=411-525 trajectories). ( d ) Table of the distributions of each minimal distance interaction type, including indirect (50-100 nm), microdomain (100-500 nm) and remote (500< nm), between GluR2-AMPAR and GPER1 on PC12 neurons after vehicle or E2 treatment (the minimal distance is expressed as the median ± 25-75% (in brackets)).

Article Snippet: Before single-molecule imaging, PC12 neurons were incubated in dRPMI with ATTO 488-labeled antibodies directed against the extracellular N-terminal domain of rat GluR2 (1:100, Alomone Labs) or against the extracellular N-terminal domain of rat mGluR1 (1:100, Alomone Labs) at 37°C for 6 minutes.

Techniques: Gel Electrophoresis, Expressing, Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Single-molecule analysis reveals the rapid effect of estradiol on the surface movement of AMPAR in live neurons

doi: 10.1101/602813

Figure Lengend Snippet: The role of the cortical actin network in the rapid effect of E2. ( a ) The actin polymerization blocker latrunculin A (latA) decreased the dynamics of the cortical actin network in CHO cells (as % of control: 51 ± 11) (for the methods of analysis of cortical actin network dynamics, see Supplementary Methods). Neither 100 nM nor 100 pM E2 affected actin dynamics (as % of control: 100 pM E2 = 108 ± 30; 100 nM E2 = 93 ± 14) (n=5-6 cells per group) ( b ) Confocal images depicting the Alexa Fluor 488 phalloidin-labeled cortical actin network in PC12 neurons after vehicle or 10-minute 1 µM latA treatment in PC12 neurons. The bar graph shows the decreased integrated density of the cortical actin network after latA exposure (right, n=3 cells per group (3 regions of interest per cell)). Scale bar: 5 µm. ( c ). LatA treatment increased the diffusion coefficient (D) of GluR2-AMPA molecules on somas with no effect on the molecules on neurites (expressed as the median ± 25-75%, n = 307-507 trajectories). ( d ) On somas, 100 pM E2 decreased the D values of GluR2-AMPAR molecules. This effect was blocked in the presence of latA. Ineffectiveness of E2 action on neurite was not affected by latA treatment (expressed as the median ± 25-75%, n = 119-205 trajectories). **p<0.01, ***p<0.001

Article Snippet: Before single-molecule imaging, PC12 neurons were incubated in dRPMI with ATTO 488-labeled antibodies directed against the extracellular N-terminal domain of rat GluR2 (1:100, Alomone Labs) or against the extracellular N-terminal domain of rat mGluR1 (1:100, Alomone Labs) at 37°C for 6 minutes.

Techniques: Control, Analysis, Confocal, Labeling, Diffusion-based Assay

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, Saline, Control

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, Control

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