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

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: The following antibodies were used: mouse monoclonal anti-TG2 (IA12), anti-FLOT-2 (BD Biosciences, Wokingham, United Kingdom), anti-PSD-95 (Neuromab, Davis, C, USA), anti-TOM20 (Santa Cruz, Dallas, Texas, USA) and polyclonal rabbit anti-TG2 (ab421) (Abcam), anti-β-tubulin (Abcam), anti-ALIX (Covalab, Bron, France), anti-NR2B (Alomone, Jerusalem, Israel), anti-VGAT (Synaptic systems, Goettingen, Germany), anti-VGLUT1 (Synaptic systems), anti-GFAP (GeneTex).

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

Journal: Translational Psychiatry

Article Title: Alteration of NMDA receptor trafficking as a cellular hallmark of psychosis

doi: 10.1038/s41398-021-01549-7

Figure Lengend Snippet: a Schematic description of the experimental workflow. CSF from patients with schizophrenic spectrum disorders (SCZSD, n = 12), affective spectrum disorders (AffectSD, n = 9) subarachnoid hemorrhage (SAH, n = 9 patients), brain polytrauma ( n = 3), hemophagocytic lymphohistiocytosis (HLH, n = 1), were collected. Then, single nanoparticle (QDot) tracking of GluN2B-NMDAR was performed onto cultured hippocampal neurons exposed to CSF. b Representative trajectories (50 ms acquisition) of surface GluN2B-NMDAR-QD complexes in dendritic spines of neurons exposed to various CSFs. Scale bar = 300 nm. c Diffusion coefficient (µm 2 /s) of synaptic GluN2B-NMDAR values after exposure for 15–30 min to patients’ CSF. Each plotted dot corresponds to the median diffusion coefficient value of one patient CSF. The mean and standard deviation (SD) are represented for each condition. Right panel , Comparison of GluN2B-NMDAR synaptic diffusion coefficient values after exposure for 15–30 min to patients’ CSF (Control, n = 2757 trajectories; SCZSD, n = 5849; AffectSD, n = 2410; Polytrauma, n = 825; SAB, n = 3957; *** p < 0.0001, Kruskal–Wallis followed by Dunn’s multiple comparison test). Data are represented as mean ± sem. d Cumulative distributions of GluN2B-NMDAR instantaneous diffusion coefficient (µm 2 /s) in control condition and SCZSD CSF ( p < 0.0001, Kolmogorov–Smirnov test).

Article Snippet: For x-link experiments, the control group received 1 µl of anti-rabbit alexa 568 (control IgG, 1/5), while the GluN2B-x-link group received 1 mg of polyclonal rabbit anti-GluN2B subunit (Alomone Labs).

Techniques: Cell Culture, Diffusion-based Assay, Standard Deviation

Journal: Translational Psychiatry

Article Title: Alteration of NMDA receptor trafficking as a cellular hallmark of psychosis

doi: 10.1038/s41398-021-01549-7

Figure Lengend Snippet: a Schematic description of the experimental workflow: we compared the GluN2B-NMDAR surface dynamics onto neurons in which we downregulated either IL1RAPL1 or DISC1. b Representative trajectories (50 ms acquisition) of surface GluN2B-NMDAR-QD complexes on neurons transfected with either a scrambled siRNA (DISC1 Scr) or DISC1 siRNA (DISC1 Kd). Scale bar = 300 nm. Below , Cumulative distributions of synaptic GluN2B-NMDAR instantaneous diffusion coefficient (µm 2 /s) in DISC1 Scr and DISC1 Kd (Scr, n = 976 trajectories; Kd, n = 662; *** p < 0.0001, Kruskal–Wallis followed by Dunn’s multiple comparison test). c Representative trajectories (50 ms acquisition) of surface GluN2B-NMDAR-QD complexes on neurons from IIL1RAPL1 wild-type or KO mice. Scale bar = 300 nm. Below , Cumulative distributions of synaptic GluN2B-NMDAR instantaneous diffusion coefficient (µm 2 /s) in WT and KO conditions (WT, n = 1430 trajectories; KO, n = 1097; p > 0.05).

Article Snippet: For x-link experiments, the control group received 1 µl of anti-rabbit alexa 568 (control IgG, 1/5), while the GluN2B-x-link group received 1 mg of polyclonal rabbit anti-GluN2B subunit (Alomone Labs).

Techniques: Transfection, Diffusion-based Assay

Journal: Translational Psychiatry

Article Title: Alteration of NMDA receptor trafficking as a cellular hallmark of psychosis

doi: 10.1038/s41398-021-01549-7

Figure Lengend Snippet: a Schematic description of the experimental workflow. Cultured hippocampal networks were made from rat pups. Examples of immuncytochemical staining of neurons (NeuN, green), nucleus (DAPI, blue), and glial cells (GFAP, red) in cultured network at 10 days in vitro from control or MAM-exposed pups. b Representative trajectories (50 ms acquisition) of surface GluN2B-NMDAR-QD complexes on neurons from control or MAM-exposed pups at three developmental stages: days in vitro 6–10 (D8), days in vitro 10–15 (D12), days in vitro 15–22 (D20). Scale bars = 5 µm/300 nm (left/right). Right, Comparison of GluN2B-NMDAR diffusion coefficient control or MAM-exposed conditions (D8 cont, n = 1916 trajectories; D8 MAM, n = 1220; D12 cont, n = 2055; D12 MAM, n = 946; D20 cont, n = 1288; D20 MAM, n = 1317; ** p < 0.01, Mann–Whitney test). Data are represented as mean ± sem. c Surface immunostaining of GluN2B-NMDAR onto neurons from control or MAM-exposed pups (synapses were detected with Homer1c-GFP). Scale bars = 5/1 µm (left/right). d Comparison of the GluN2B, GluN2A, GluN3A-NMDAR cluster areas between conditions (GluN2B synaptic cont, n = 2144 clusters; GluN2B synaptic MAM, n = 2468, *** p < 0.001, Student’s t -test; GluN2B extrasynaptic cont, n = 1733; GluN2B extrasynaptic MAM, n = 1421, ** p < 0.01, Student’s t -test; GluN2A cont, n = 56; GluN2A MAM, n = 41; GluN3A cont, n = 59; GluN3A MAM, n = 48).

Article Snippet: For x-link experiments, the control group received 1 µl of anti-rabbit alexa 568 (control IgG, 1/5), while the GluN2B-x-link group received 1 mg of polyclonal rabbit anti-GluN2B subunit (Alomone Labs).

Techniques: Cell Culture, Staining, In Vitro, Diffusion-based Assay, MANN-WHITNEY, Immunostaining

Journal: Translational Psychiatry

Article Title: Alteration of NMDA receptor trafficking as a cellular hallmark of psychosis

doi: 10.1038/s41398-021-01549-7

Figure Lengend Snippet: a Spontaneous AMPAR and NMDAR-mediated EPSCs recorded in whole-cell configuration at −70 and +40 mV, respectively, in the CA1 of hippocampal slices from control and MAM-exposed animals. Insets show superimposed examples of spontaneous AMPAR and NMDAR-mediated responses as well as evoked NMDAR-mediated currents. b Spontaneous ( n (cont/MAM) = 14/20; p < 0.05) as well as evoked ( n (cont/MAM) = 16/16; p < 0.05) NMDAR-mediated currents are significantly larger in MAM-exposed animals. Also, the Tau decay was increased by ~25% ( n (cont/MAM) = 8/16; p < 0.05), which is consistent with an increased synaptic expression of GluN2B. c Tonic NMDA currents were recorded as the subtracted holding current at a depolarized potential of +40 mV before and after the addition of APV. These tonic NMDA currents were significantly decreased in MAM-exposed animals ( n (cont/MAM) = 10/10; p < 0.05), as revealed by a much smaller decrease in holding current with APV. d Field excitatory postsynaptic potential (fEPSP) recorded in CA1 pyramidal cell layer after stimulation of hippocampal Schaffer collaterals. Active input displays a significant postsynaptic potentiation (LTP) following high-frequency stimulation at P10 (control), not visible in non-stimulated input ( n = 6; p < 0.05). e When comparing the response to high-frequency stimulation in control and MAM-exposed animals there was a clear deficit in the magnitude of the LTP in MAM-exposed rats between P8 and P12. This deficit was transient and disappeared with age such that the LTP recovered in older animals.

Article Snippet: For x-link experiments, the control group received 1 µl of anti-rabbit alexa 568 (control IgG, 1/5), while the GluN2B-x-link group received 1 mg of polyclonal rabbit anti-GluN2B subunit (Alomone Labs).

Techniques: Expressing

Journal: Translational Psychiatry

Article Title: Alteration of NMDA receptor trafficking as a cellular hallmark of psychosis

doi: 10.1038/s41398-021-01549-7

Figure Lengend Snippet: a Schematic description of the experimental workflow: control of MAM-exposed pups received intracerebral injections during the second postnatal week of NMDAR surface diffusion modulators. b Representative trajectories (50 ms acquisition) of surface GluN2B-NMDAR on hippocampal neurons exposed to TAT-non sense (TAT-NS, 10 µM), TAT-2B (10 µM), control IgG (10 µg/ml), or IgG direct against extracellular epitope of the GluN2B-NMDAR (10 µg/ml). Scale bars = 500 nm (left/right). c The startle response, i.e., pre-pulse inhibition (PPI), was measured at P60 and compared between conditions ( n = 10–16 rats per group; * P < 0.05, ** P < 0.01, ANOVA 1 followed by Newman–Keuls multiple comparisons test).

Article Snippet: For x-link experiments, the control group received 1 µl of anti-rabbit alexa 568 (control IgG, 1/5), while the GluN2B-x-link group received 1 mg of polyclonal rabbit anti-GluN2B subunit (Alomone Labs).

Techniques: Diffusion-based Assay, Inhibition