Journal: PLoS ONE

Article Title: Chronic Zinc Exposure Decreases the Surface Expression of NR2A-Containing NMDA Receptors in Cultured Hippocampal Neurons

doi: 10.1371/journal.pone.0046012

Figure Lengend Snippet: (A) Representative images of clustering of NR2B in control, NBQX+nimodipine and Zn+NBQX+nimodipine groups. Higher magnification views are shown of the dendritic branches studded with numerous clusters enclosed in white boxes. Scale bar, 50 µm. (B) and (C) show the quantification of (A). The numbers of NR2B clusters were 25.1±1.0 (control, n = 37), 25.5±1.0 (NBQX+nimodipine, n = 36), and 25.7±1.0 (Zn+NBQX+nimodipine, n = 37) per 100 µm dendrite length. The mean intensity of NR2B clusters was 122.4±3.3 (control, n = 25), 133.0±4.5 (NBQX+nimodipine, n = 25) and 131.3±3.6 (Zn+NBQX+nimodipine, n = 23). (D) Representative NR2B currents in response to co-application with the agonist plus NVP-AAM077. (E) Statistics of NR2B currents. Averaged peak currents were 342.7±71.7 pA (control, n = 38), 327.0±67.3 pA (NBQX+nimodipine, n = 20) and 320.0±48.5 pA (Zn+NBQX+nimodipine, n = 20).

Article Snippet: Rabbit polyclonal NR2B antibody was from Alomone Labs (Jerusalem, Israel).

Techniques:

Journal: PLoS ONE

Article Title: Chronic Zinc Exposure Decreases the Surface Expression of NR2A-Containing NMDA Receptors in Cultured Hippocampal Neurons

doi: 10.1371/journal.pone.0046012

Figure Lengend Snippet: (A) Hippocampal neurons derived from control, NBQX+nimodipine and Zn+NBQX+nimodipine groups were incubated in 35 µg/ml CHX. Total expressions of NR1, NR2A and NR2B were measured at 0 hr, 12 hr or 18 hr in the CHX treatment. (B) shows the quantification of (A). (C) Surface NR1, NR2A and NR2B derived from cultured hippocampal neurons were isolated by biotinylation assay and detected by anti-NR1, anti-NR2A and anti-NR2B antibodies. Surface NR1 and NR2A, but not NR2B, were notably lower in the Zn+NBQX+nimodipine group, while total NR1, NR2A and NR2B did not change. (D) Quantifications of (C). Percentage changes of surface signal intensities versus control were: NR1, 97.6±13.8 (NBQX+nimodipine) and 69.4±8.0 (Zn+NBQX+nimodipine); NR2A, 87.7±4.9 (NBQX+nimodipine) and 57.0±14.3 (Zn+NBQX+nimodipine); and NR2B, 99.0±19.0 (NBQX+nimodipine) and 101.0±21.8 (Zn+NBQX+nimodipine). All experiments were performed at least 3 times (N > = 3). *, P<0.05.

Article Snippet: Rabbit polyclonal NR2B antibody was from Alomone Labs (Jerusalem, Israel).

Techniques: Derivative Assay, Incubation, Cell Culture, Isolation, Cell Surface Biotinylation Assay

Journal: PLoS ONE

Article Title: Chronic Zinc Exposure Decreases the Surface Expression of NR2A-Containing NMDA Receptors in Cultured Hippocampal Neurons

doi: 10.1371/journal.pone.0046012

Figure Lengend Snippet: (A) Lysates were immunoprecipitated with anti-PSD-95 antibody in control, NBQX+nimodipine and Zn+NBQX+nimodipine groups. The immunoprecipitates were probed with the anti-NR2A (NR2A), anti-NR1 (NR1), anti-NR2B (NR2B), anti-Src (Src) and anti-Fyn (Fyn) antibodies. The Input lane was loaded with total protein (20 µg) derived from cultured hippocampal neurons. (B) Supernatant samples were immunoprecipitated with anti-PSD-95 antibody in different groups, which showed the clearance of PSD95 after Co-IP experiment. All experiments were performed at least 3 times (N > = 3) and the summarized data are shown in (C) and (D). Y axes in (C) and (D) represent the percentage change of each co-precipitated protein (labeled on X axes) relative to the corresponding control. *, P<0.05.

Article Snippet: Rabbit polyclonal NR2B antibody was from Alomone Labs (Jerusalem, Israel).

Techniques: Immunoprecipitation, Derivative Assay, Cell Culture, Co-Immunoprecipitation Assay, Labeling

Journal: bioRxiv

Article Title: Astrocytic extracellular vesicles modulate neuronal calcium homeostasis via transglutaminase-2

doi: 10.1101/2021.09.30.462507

Figure Lengend Snippet: A) Immunofluorescence staining of primary astrocytes. Cells were fixed in 4% paraformaldehyde - 4 % sucrose (w/v), permeabilized (left panels) or non-permeabilized (right panel) and stained with anti-TG2 IA12 (green), DAPI (blue) and astrocytic marker anti-GFAP (red). Coverslips were visualised by laser scanning Leica SP5 confocal microscope using 63X oil immersion objective. Successive serial optical sections (1 µm) were recorded over 8 µm planes. Scale bar 20 µm. TG2 intensity was calculated by Leica software, divided by number of nuclei, and normalised to permeabilized values. Data is expressed as mean ± SD (N=3, Mann-Whitney test: p=NS). B) Neurons at 12 DIV were fixed and permeabilized (left panel) or non-permeabilized (right panel) and stained with anti-TG2 IA12 (green), anti-β-TUB or anti-NR2B (red) antibodies and DAPI (blue). Scale bar 10 µm. TG2 intensity was calculated as described in A (N=3, Mann-Whitney test: *p<0.05). C) Neurons at 22 Days In Vitro (DIV) were fixed, permeabilized and stained with anti-TG2 IA12 (green), anti-SHANK2 (red) and anti-VGLUT1 (blue) antibodies (N=3). Coverslips were visualised as described in A. Scale bar 20 µm (low magnification) and 5 µm (high magnification). D) Crude synaptosomes were isolated from adult mouse brain and probed with pre- and post-synaptic markers and for astrocyte marker GFAP. B-tubulin was used as loading control. TG2 is enriched in the synaptic fraction (Syn) compared to Low Speed Supernatant (LSS) representing the total lysate. Data is expressed as mean ± SD (N=4 independent experiments, Mann-Whitney test: *p<0.05).

Article Snippet: Cells were fixed in 4% paraformaldehyde - 4 % sucrose (w/v) and immunofluorescence staining was performed using the following antibodies: mouse monoclonal anti-TG2 (IA12 – Tim Johnson, University of Sheffield ( )), guinea pig anti-VGLUT1 (Synaptic System, Goettingen, Germany), rabbit anti-GFAP (Dako, Agilent, Santa Clara, CA, USA), rabbit anti-Shank2 (Synaptic System), rabbit anti-Fibronectin (Sigma-Aldrich), rabbit anti-β-tubulin (Sigma-Aldrich) and rabbit anti-NR2B (Alomone, Jerusalem, Israel).

Techniques: Immunofluorescence, Staining, Marker, Microscopy, Software, MANN-WHITNEY, In Vitro, Isolation