Journal: International Journal of Molecular Sciences

Article Title: Metabotropic Glutamate Receptor 3 Expression During Liver Disease Progression: Association with Inflammation and Cell Viability in Hepatocellular Carcinoma

doi: 10.3390/ijms27093878

Figure Lengend Snippet: Liver mGluR3 expression and correlation of its mRNA levels with inflammation related-cytokines. Increased mGluR3 ( Grm3 ) mRNA expression and a positive correlation with Il-6 and Tgfb were observed in HCC. ( A ) Grm3 expression in perfused livers of control, fibrosis, cirrhosis, and HCC rats was analyzed by RT-qPCR. In HCC, DEN-PZ indicates the peritumoral zone. The graph represents mean ± SEM ( n = 5–12). Values were normalized to Rps18 gene expression. * p < 0.05 by one-way ANOVA and Dunn’s post hoc test. ( B ) The identity of the Grm3 qPCR product was confirmed by Sanger sequencing, and its size was verified by agarose gel electrophoresis. Exons of Grm3 and the position of the oligonucleotides used are shown. ( C ) Spearman’s correlation plots between Grm3 and inflammation related-cytokines in HCC tissue samples with significant coefficients are shown. ( D , E ) Detection of the mGluR3 protein in different rodent tissues (r-rat, m-mouse). A band of the expected weight of ~98 kDa was detected in all tissues analyzed using anti-mGluR3 from ( D ) Abcam ® and ( E ) Alomone ® . An additional band (~103 kDa) was observed in the liver and isolated hepatocytes, using Abcam ® antibody (HCC and control liver tissues obtained from the DEN model).

Article Snippet: Diethylnitrosamine (DEN), (N0756), glutamate assay kit (MAK330), Minimum Essential Medium Eagle (MEM) (M0643), F12 Ham Kaighn’s Modification (F12K) (N3520), LY354740 (L1045), glutamate (L-glutamic acid monosodium salt, G5889), propidium iodide (P4170) were purchased from Sigma-Aldrich, St. Louis, MO, USA; Dulbecco’s Modified Eagle Medium (DMEM) low glucose from Gibco (Waltham, MA, USA); polyclonal rabbit anti-mGluR3 antibody (#AGC-012), mGluR3 blocking peptide (BLP-GC012) from Alomone (Jerusalem, Israel); polyclonal rabbit anti-mGluR3 antibody (ab140741), biotinylated goat anti-rabbit IgG antibody (ab6720), peroxidase–conjugated donkey anti-rabbit IgG antibody (AB97061) from Abcam (Cambridge, UK); monoclonal mouse anti-cAMP antibody (MAB2146) from R&D (Minneapolis, MN, USA); monoclonal rabbit anti-GAPDH (14C10), monoclonal rabbit b-actin (D6A8) from Cell Signaling Technology (Danvers, MA, USA); forskolin (1099) from Tocris (Minneapolis, MN, USA); RevertAid First Strand cDNA Synthesis Kit, Maxima SYBR Green qPCR Master Mix from Thermo Scientific (Waltham, MA, USA); TRIzolTM reagent, Alexa Fluor-488 donkey anti-rabbit IgG (A-21206), Alexa Fluor-488 goat anti-mouse IgG (H + L) (A-11001) from Invitrogen (Waltham, MA, USA); Vectashield ® (H1000), Vectastain Elite ABC-HRP kit (PK-6100), DAB Substrate Kit, Peroxidase (HRP), (3,3′-diaminobenzidine) (SK-4100) from Vector Laboratories (Newark, CA, USA); Direct-zolTM Miniprep RNA kit from Zymo Research (R2050) (Irving, CA, USA); AP conjugate substrate kit (1706432), protein assay dye (5000006) from Bio-Rad (Hercules, CA, USA).

Techniques: Expressing, Control, Quantitative RT-PCR, Gene Expression, Sequencing, Agarose Gel Electrophoresis, Isolation

Journal: International Journal of Molecular Sciences

Article Title: Metabotropic Glutamate Receptor 3 Expression During Liver Disease Progression: Association with Inflammation and Cell Viability in Hepatocellular Carcinoma

doi: 10.3390/ijms27093878

Figure Lengend Snippet: Liver immunodetection of mGluR3 during liver pathology progression in DEN-treated rats. ( A ) Representative images of mGluR3 staining (brown) and toluidine blue counterstaining of liver tissue from normal (control), fibrosis, cirrhosis, and HCC. The absence of primary antibody ( w / o ) or specific blocking peptide (BP) pre-incubated with anti-mGluR3 was used as a negative control. To the left of the dashed line, a dysplastic lesion associated with cirrhosis and a neoplastic lesion related to HCC are observed. Scale bar, 200 μm (10×) or 50 μm (40×). ( B ) Quantitative analysis was performed in ImageJ software. The graph represents quantification of mGluR3 by area in 10 high-power fields (40×). Data are expressed as mean ± SEM of three independent experiments, * p < 0.01 by t -test.

Article Snippet: Diethylnitrosamine (DEN), (N0756), glutamate assay kit (MAK330), Minimum Essential Medium Eagle (MEM) (M0643), F12 Ham Kaighn’s Modification (F12K) (N3520), LY354740 (L1045), glutamate (L-glutamic acid monosodium salt, G5889), propidium iodide (P4170) were purchased from Sigma-Aldrich, St. Louis, MO, USA; Dulbecco’s Modified Eagle Medium (DMEM) low glucose from Gibco (Waltham, MA, USA); polyclonal rabbit anti-mGluR3 antibody (#AGC-012), mGluR3 blocking peptide (BLP-GC012) from Alomone (Jerusalem, Israel); polyclonal rabbit anti-mGluR3 antibody (ab140741), biotinylated goat anti-rabbit IgG antibody (ab6720), peroxidase–conjugated donkey anti-rabbit IgG antibody (AB97061) from Abcam (Cambridge, UK); monoclonal mouse anti-cAMP antibody (MAB2146) from R&D (Minneapolis, MN, USA); monoclonal rabbit anti-GAPDH (14C10), monoclonal rabbit b-actin (D6A8) from Cell Signaling Technology (Danvers, MA, USA); forskolin (1099) from Tocris (Minneapolis, MN, USA); RevertAid First Strand cDNA Synthesis Kit, Maxima SYBR Green qPCR Master Mix from Thermo Scientific (Waltham, MA, USA); TRIzolTM reagent, Alexa Fluor-488 donkey anti-rabbit IgG (A-21206), Alexa Fluor-488 goat anti-mouse IgG (H + L) (A-11001) from Invitrogen (Waltham, MA, USA); Vectashield ® (H1000), Vectastain Elite ABC-HRP kit (PK-6100), DAB Substrate Kit, Peroxidase (HRP), (3,3′-diaminobenzidine) (SK-4100) from Vector Laboratories (Newark, CA, USA); Direct-zolTM Miniprep RNA kit from Zymo Research (R2050) (Irving, CA, USA); AP conjugate substrate kit (1706432), protein assay dye (5000006) from Bio-Rad (Hercules, CA, USA).

Techniques: Immunodetection, Staining, Control, Blocking Assay, Incubation, Negative Control, Software

Journal: International Journal of Molecular Sciences

Article Title: Metabotropic Glutamate Receptor 3 Expression During Liver Disease Progression: Association with Inflammation and Cell Viability in Hepatocellular Carcinoma

doi: 10.3390/ijms27093878

Figure Lengend Snippet: Cellular immunodetection of mGluR3 in HCC livers of DEN-treated rats. ( Upper panel ) Representative images of mGluR3 staining (brown) and toluidine blue counterstaining of liver tissue from normal (control), fibrosis, cirrhosis, and HCC. Scale bar, 30 μm (100×). Arrows indicate plasma or nuclear membrane, and arrowheads indicate the cytoplasmic location of mGluR3. ( Lower panel ) Zoomed-in areas are shown on the right. The control group shows a light positive signal for mGluR3 in intracellular locations, while the cirrhosis group exhibits a strong positive signal. Identification tags: A: artery, V: vein, O: hyperplastic oval cells, BD: bile ducts, KC: Kupffer cells, EC: endothelial cells.

Article Snippet: Diethylnitrosamine (DEN), (N0756), glutamate assay kit (MAK330), Minimum Essential Medium Eagle (MEM) (M0643), F12 Ham Kaighn’s Modification (F12K) (N3520), LY354740 (L1045), glutamate (L-glutamic acid monosodium salt, G5889), propidium iodide (P4170) were purchased from Sigma-Aldrich, St. Louis, MO, USA; Dulbecco’s Modified Eagle Medium (DMEM) low glucose from Gibco (Waltham, MA, USA); polyclonal rabbit anti-mGluR3 antibody (#AGC-012), mGluR3 blocking peptide (BLP-GC012) from Alomone (Jerusalem, Israel); polyclonal rabbit anti-mGluR3 antibody (ab140741), biotinylated goat anti-rabbit IgG antibody (ab6720), peroxidase–conjugated donkey anti-rabbit IgG antibody (AB97061) from Abcam (Cambridge, UK); monoclonal mouse anti-cAMP antibody (MAB2146) from R&D (Minneapolis, MN, USA); monoclonal rabbit anti-GAPDH (14C10), monoclonal rabbit b-actin (D6A8) from Cell Signaling Technology (Danvers, MA, USA); forskolin (1099) from Tocris (Minneapolis, MN, USA); RevertAid First Strand cDNA Synthesis Kit, Maxima SYBR Green qPCR Master Mix from Thermo Scientific (Waltham, MA, USA); TRIzolTM reagent, Alexa Fluor-488 donkey anti-rabbit IgG (A-21206), Alexa Fluor-488 goat anti-mouse IgG (H + L) (A-11001) from Invitrogen (Waltham, MA, USA); Vectashield ® (H1000), Vectastain Elite ABC-HRP kit (PK-6100), DAB Substrate Kit, Peroxidase (HRP), (3,3′-diaminobenzidine) (SK-4100) from Vector Laboratories (Newark, CA, USA); Direct-zolTM Miniprep RNA kit from Zymo Research (R2050) (Irving, CA, USA); AP conjugate substrate kit (1706432), protein assay dye (5000006) from Bio-Rad (Hercules, CA, USA).

Techniques: Immunodetection, Staining, Control, Clinical Proteomics, Membrane

Journal: International Journal of Molecular Sciences

Article Title: Metabotropic Glutamate Receptor 3 Expression During Liver Disease Progression: Association with Inflammation and Cell Viability in Hepatocellular Carcinoma

doi: 10.3390/ijms27093878

Figure Lengend Snippet: Immunodetection of mGluR3 in liver cell lines. Immunofluorescence staining for mGluR3 (green) and nuclei (red, propidium iodide) in C9 (rat normal hepatocytes) and HepG2 (human HCC cells); the merge of both channels is displayed. Immunostaining with anti-mGluR3 ( A ) Abcam and ( B ) Allomone antibodies is shown. Scale bar = 50 μm (25×) and 20 μm (crop). Crops of representative cells (white squares) are shown on the right. Negative controls in the absence of a primary antibody are shown as inserts (red squares). Quantitative analysis was performed in ImageJ software. Graphs represent the percentage of mGluR3-positive cells, and the average fluorescence intensity of mGluR3 was measured in 9–20 fields. Data are expressed as mean ± SEM of three independent cultures, ** p < 0.01.

Article Snippet: Diethylnitrosamine (DEN), (N0756), glutamate assay kit (MAK330), Minimum Essential Medium Eagle (MEM) (M0643), F12 Ham Kaighn’s Modification (F12K) (N3520), LY354740 (L1045), glutamate (L-glutamic acid monosodium salt, G5889), propidium iodide (P4170) were purchased from Sigma-Aldrich, St. Louis, MO, USA; Dulbecco’s Modified Eagle Medium (DMEM) low glucose from Gibco (Waltham, MA, USA); polyclonal rabbit anti-mGluR3 antibody (#AGC-012), mGluR3 blocking peptide (BLP-GC012) from Alomone (Jerusalem, Israel); polyclonal rabbit anti-mGluR3 antibody (ab140741), biotinylated goat anti-rabbit IgG antibody (ab6720), peroxidase–conjugated donkey anti-rabbit IgG antibody (AB97061) from Abcam (Cambridge, UK); monoclonal mouse anti-cAMP antibody (MAB2146) from R&D (Minneapolis, MN, USA); monoclonal rabbit anti-GAPDH (14C10), monoclonal rabbit b-actin (D6A8) from Cell Signaling Technology (Danvers, MA, USA); forskolin (1099) from Tocris (Minneapolis, MN, USA); RevertAid First Strand cDNA Synthesis Kit, Maxima SYBR Green qPCR Master Mix from Thermo Scientific (Waltham, MA, USA); TRIzolTM reagent, Alexa Fluor-488 donkey anti-rabbit IgG (A-21206), Alexa Fluor-488 goat anti-mouse IgG (H + L) (A-11001) from Invitrogen (Waltham, MA, USA); Vectashield ® (H1000), Vectastain Elite ABC-HRP kit (PK-6100), DAB Substrate Kit, Peroxidase (HRP), (3,3′-diaminobenzidine) (SK-4100) from Vector Laboratories (Newark, CA, USA); Direct-zolTM Miniprep RNA kit from Zymo Research (R2050) (Irving, CA, USA); AP conjugate substrate kit (1706432), protein assay dye (5000006) from Bio-Rad (Hercules, CA, USA).

Techniques: Immunodetection, Immunofluorescence, Staining, Immunostaining, Software, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Metabotropic Glutamate Receptor 3 Expression During Liver Disease Progression: Association with Inflammation and Cell Viability in Hepatocellular Carcinoma

doi: 10.3390/ijms27093878

Figure Lengend Snippet: Effect of mGluR3 activation by glutamate or LY354740 on cAMP content induced by forskolin in HepG2 cells. ( A ) Immunofluorescence staining for cAMP (green) and nuclei (red, propidium iodide) in HepG2 cells; the merge of both channels is displayed. All cells displayed a positive cAMP signal, with different levels depending on the treatment. cAMP was enhanced by the adenylate cyclase activator forskolin (Fk, 10 μM) that was inhibited by glutamate (glu, 10 μM) or the mGluR3 selective agonist LY354740 (LY, 40 nM). Scale bar = 50 μm (25×) or 20 μm (crop). ( B ) Quantitative analysis of the number of positive cells and fluorescence intensity of cAMP measured in 10 fields. Data are expressed as mean ± SEM of three independent cultures. ** p < 0.01.

Article Snippet: Diethylnitrosamine (DEN), (N0756), glutamate assay kit (MAK330), Minimum Essential Medium Eagle (MEM) (M0643), F12 Ham Kaighn’s Modification (F12K) (N3520), LY354740 (L1045), glutamate (L-glutamic acid monosodium salt, G5889), propidium iodide (P4170) were purchased from Sigma-Aldrich, St. Louis, MO, USA; Dulbecco’s Modified Eagle Medium (DMEM) low glucose from Gibco (Waltham, MA, USA); polyclonal rabbit anti-mGluR3 antibody (#AGC-012), mGluR3 blocking peptide (BLP-GC012) from Alomone (Jerusalem, Israel); polyclonal rabbit anti-mGluR3 antibody (ab140741), biotinylated goat anti-rabbit IgG antibody (ab6720), peroxidase–conjugated donkey anti-rabbit IgG antibody (AB97061) from Abcam (Cambridge, UK); monoclonal mouse anti-cAMP antibody (MAB2146) from R&D (Minneapolis, MN, USA); monoclonal rabbit anti-GAPDH (14C10), monoclonal rabbit b-actin (D6A8) from Cell Signaling Technology (Danvers, MA, USA); forskolin (1099) from Tocris (Minneapolis, MN, USA); RevertAid First Strand cDNA Synthesis Kit, Maxima SYBR Green qPCR Master Mix from Thermo Scientific (Waltham, MA, USA); TRIzolTM reagent, Alexa Fluor-488 donkey anti-rabbit IgG (A-21206), Alexa Fluor-488 goat anti-mouse IgG (H + L) (A-11001) from Invitrogen (Waltham, MA, USA); Vectashield ® (H1000), Vectastain Elite ABC-HRP kit (PK-6100), DAB Substrate Kit, Peroxidase (HRP), (3,3′-diaminobenzidine) (SK-4100) from Vector Laboratories (Newark, CA, USA); Direct-zolTM Miniprep RNA kit from Zymo Research (R2050) (Irving, CA, USA); AP conjugate substrate kit (1706432), protein assay dye (5000006) from Bio-Rad (Hercules, CA, USA).

Techniques: Activation Assay, Immunofluorescence, Staining, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Metabotropic Glutamate Receptor 3 Expression During Liver Disease Progression: Association with Inflammation and Cell Viability in Hepatocellular Carcinoma

doi: 10.3390/ijms27093878

Figure Lengend Snippet: Time-course effect of mGluR3 activation by glutamate or LY354740 on viability in cultured HepG2 cells. ( A ) Time-course of viable cells, and ( B ) dead cells under treatment with or without mGluR3 agonists, visualized by trypan blue staining. The activation of mGluR3 by glutamate or LY354740 significantly influenced the viability of cultured HepG2 cells over time. Data are expressed as mean ± SD of five independent experiments; * p < 0.01; ** p < 0.05 vs. control by one-way ANOVA and Dunn’s post hoc test.

Article Snippet: Diethylnitrosamine (DEN), (N0756), glutamate assay kit (MAK330), Minimum Essential Medium Eagle (MEM) (M0643), F12 Ham Kaighn’s Modification (F12K) (N3520), LY354740 (L1045), glutamate (L-glutamic acid monosodium salt, G5889), propidium iodide (P4170) were purchased from Sigma-Aldrich, St. Louis, MO, USA; Dulbecco’s Modified Eagle Medium (DMEM) low glucose from Gibco (Waltham, MA, USA); polyclonal rabbit anti-mGluR3 antibody (#AGC-012), mGluR3 blocking peptide (BLP-GC012) from Alomone (Jerusalem, Israel); polyclonal rabbit anti-mGluR3 antibody (ab140741), biotinylated goat anti-rabbit IgG antibody (ab6720), peroxidase–conjugated donkey anti-rabbit IgG antibody (AB97061) from Abcam (Cambridge, UK); monoclonal mouse anti-cAMP antibody (MAB2146) from R&D (Minneapolis, MN, USA); monoclonal rabbit anti-GAPDH (14C10), monoclonal rabbit b-actin (D6A8) from Cell Signaling Technology (Danvers, MA, USA); forskolin (1099) from Tocris (Minneapolis, MN, USA); RevertAid First Strand cDNA Synthesis Kit, Maxima SYBR Green qPCR Master Mix from Thermo Scientific (Waltham, MA, USA); TRIzolTM reagent, Alexa Fluor-488 donkey anti-rabbit IgG (A-21206), Alexa Fluor-488 goat anti-mouse IgG (H + L) (A-11001) from Invitrogen (Waltham, MA, USA); Vectashield ® (H1000), Vectastain Elite ABC-HRP kit (PK-6100), DAB Substrate Kit, Peroxidase (HRP), (3,3′-diaminobenzidine) (SK-4100) from Vector Laboratories (Newark, CA, USA); Direct-zolTM Miniprep RNA kit from Zymo Research (R2050) (Irving, CA, USA); AP conjugate substrate kit (1706432), protein assay dye (5000006) from Bio-Rad (Hercules, CA, USA).

Techniques: Activation Assay, Cell Culture, Staining, Control

Journal: Cell reports

Article Title: GluN3A subunit tunes NMDA receptor synaptic trafficking and content during postnatal brain development

doi: 10.1016/j.celrep.2023.112477

Figure Lengend Snippet: (A) Surface GluN3A-NMDARs were detected using a quantum dot (QD)-antibody complex directed against the extracellular GFP tag, allowing high accuracy detection. Left: schematic showing single-particle tracking of surface GFP-GluN3A using QDs. Right: representative summed trajectories of GluN3A-QDs (red) acquired over a period of 30 s at a 20-Hz rate in a young (9 DIV) hippocampal neuron. Scale bar, 10 μm. (B) Plot of the mean square displacement (MSD) vs. time for surface GluN3A-NMDARs (n = 143 trajectories, 44 fields from at least four different cultures) in young (7–9 DIV) neurons; dotted line is the behavior predicted for a freely diffusive receptor. (C) Representative image of a dendritic field from a hippocampal neuron co-transfected with GFP-GluN3A and the postsynaptic marker Homer 1-DsRed (in blue). Summed QD trajectories for GluN3A-NMDARs are in red. Scale bar, 3 μm. (D) Comparative cumulative distributions of the instantaneous diffusion coefficient of synaptic (n = 560) and extrasynaptic (n = 796, 47 fields from at least four different cultures) GFP-GluN3A receptors. Here and in subsequent figures, the first data point corresponds to the percentage of immobile receptors. (E) Examples of reconstructed trajectories for transfected GFP-GluN3A, SEP-GluN2A, and SEP-GluN2B (SEP is a pH-sensitive variant of GFP). Scale bar, 1 μm. (F) Cumulative distributions of the instantaneous diffusion coefficients of synaptic GFP-GluN3A (n = 560, 47 fields from at least four different cultures), SEPGluN2A (n = 1,009), and SEP-GluN2B (n = 1,531) in 7–9 DIV neurons (p < 0.001, Kruskal-Wallis followed by Dunn’s post hoc test for all conditions). (G) Comparison of the diffusion coefficients of synaptic GFP-GluN3A (n = 560 trajectories), SEP-GluN2A (n = 1,009 trajectories), and SEP-GluN2B (n = 1,531 trajectories). ***p < 0.001, Kruskal-Wallis followed by Dunn’s post hoc test for all conditions. (H) Cumulative distributions of the instantaneous diffusion coefficients of synaptic GFP-GluN3A in control (n = 285), TTX (n = 214), or bicuculline (n = 61) (***p < 0.001, Kruskal-Wallis followed by Dunn’s post hoc test for all conditions).

Article Snippet: QD-based trajectories were considered synaptic if they colocalized with Homer 1-DsRed dendritic clusters for at least five frames. . Immunocytochemistry Neurons were fixed in 2% paraformaldehyde, 2% sucrose and stained in non-permeabilized conditions with primary antibodies against extracellular GluN2A or GluN2B (ACG-002 and ACG-003, Alomone).

Techniques: Single-particle Tracking, Transfection, Marker, Diffusion-based Assay, Variant Assay, Comparison, Control

Journal: Cell reports

Article Title: GluN3A subunit tunes NMDA receptor synaptic trafficking and content during postnatal brain development

doi: 10.1016/j.celrep.2023.112477

Figure Lengend Snippet: (A) Representative dendritic fields of 7–9 DIV hippocampal neurons transfected with Homer 1-DsRed (blue) and GFP-GluN3A, or control cells (expressing Homer 1 but not GFP-GluN3A), from the same preparation. Summed QD trajectories for native GluN2A-NMDARs and GluN2B-NMDARs are shown in red. Scale bar, 3 μm. (B) Cumulative distribution of the instantaneous diffusion coefficients of synaptic GluN2A-QDs and GluN2B-QDs in control neurons (n = 89, 15 fields from at least three different cultures, and n = 306, nine fields, from at least three different cultures, respectively), and neurons expressing GFP-GluN3A (n = 119, 22 fields from at least three different cultures, and n = 277, 18 fields, from at least three different cultures) (p < 0.0001 for GluN2A, p = 0.06 for GluN2B; Mann-Whitney test). (C) Representative dendritic fields of 7–9 DIV neurons transfected with Homer 1-DsRed (blue) and sh1185 or sh2532 as indicated. Summed QD trajectories for native GluN2A (top) and GluN2B (bottom) are shown in red. Scale bar, 3 μm. (D) Cumulative distribution of the instantaneous diffusion coefficients of synaptic GluN2A-QDs and GluN2B at 7–9 DIV in control neurons (n = 85, 46 fields from at least six different cultures, and n = 181, 41 fields from at least six different cultures, respectively), and neurons transfected with sh1185 (n = 209, 38 fields from at least six different cultures, and n = 206, 38 fields from at least six different cultures) or sh2532 (n = 240, 37 fields from at least six different cultures, and n = 158, 33 fields from at least six different cultures) (p < 0.006 for GluN2A, p = 0.08 for GluN2B; Kruskal-Wallis followed by Dunn’s multiple comparison test).

Article Snippet: QD-based trajectories were considered synaptic if they colocalized with Homer 1-DsRed dendritic clusters for at least five frames. . Immunocytochemistry Neurons were fixed in 2% paraformaldehyde, 2% sucrose and stained in non-permeabilized conditions with primary antibodies against extracellular GluN2A or GluN2B (ACG-002 and ACG-003, Alomone).

Techniques: Transfection, Control, Expressing, Diffusion-based Assay, MANN-WHITNEY, Comparison

Journal: Cell reports

Article Title: GluN3A subunit tunes NMDA receptor synaptic trafficking and content during postnatal brain development

doi: 10.1016/j.celrep.2023.112477

Figure Lengend Snippet: (A–D) Co-localization analysis of surface GluN2A- or GluN2B-NMDARs (red) with Homer1 (green) in cultured hippocampal neurons from WT and Grin3a−/− mice at young (9 DIV) and more mature stages (15–18 DIV). Representative images and quantification (mean ± SEM) are shown (n = 3 independent experiments using different cultures, p < 0.003, Student’s t test or ANOVA). Scale bar, 2 μm. In (C) and (D), Grin3a−/− neurons were transfected with GFP-GluN3A (blue) when indicated. (E) Organotypic hippocampal slice cultures from P7 rats were biolistically transfected with GFP-GluN3A subunit on DIV 3–4, and simultaneous whole-cell recordings obtained from transfected and neighboring CA1 pyramidal neurons on DIV 7. Left, sample traces of NMDAR-EPSCs from a control (black) and a GFP-GluN3A transfected cell (red). Middle, scatterplots of peak amplitudes of NMDAR-EPSCs from single pairs (open circles) and mean ± SEM (filled circle) from transfected and control cells (mean amplitude [pA]: control 169.5 ± 24.3, transfected 168.0 ± 21.0, n = 7). Dashed lines represent linear regression and 95% confidence interval. Right, NMDAR-EPSC decay times from cell pairs expressed in milliseconds as a weighted tau (τw) from paired transfected and control cells (mean decay: control 328.7 ± 17.1 ms, transfected 376.3 ± 18.0 ms, n = 7, p = 0.0002 by paired Student’s t test). (F) Percentage of block of NMDAR-EPSCs by the GluN2B selective antagonist Ro25–6981 (n = 5, p = 0.027, paired Student’s t test). (G) Schematic representation of the experimental design (left panel). Developmental time course of ifenprodil sensitivity of NMDAR-EPSCs from CA1 pyramidal neurons from WT (black circles) or Grin3a−/− mice (red circles), represented as the percentage decrease in peak current after ifenprodil. For comparison, data from Gray et al.35 of the ifenprodil sensitivity data of “pure” synaptic populations of GluN2A and GluN2B are included (green circles). (H) Western blot measurement at P15–17 of GluN2A and GluN2B subunit content in WT and Grin3a−/− mice.

Article Snippet: QD-based trajectories were considered synaptic if they colocalized with Homer 1-DsRed dendritic clusters for at least five frames. . Immunocytochemistry Neurons were fixed in 2% paraformaldehyde, 2% sucrose and stained in non-permeabilized conditions with primary antibodies against extracellular GluN2A or GluN2B (ACG-002 and ACG-003, Alomone).

Techniques: Cell Culture, Transfection, Control, Blocking Assay, Comparison, Western Blot

Journal: Cell reports

Article Title: GluN3A subunit tunes NMDA receptor synaptic trafficking and content during postnatal brain development

doi: 10.1016/j.celrep.2023.112477

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: QD-based trajectories were considered synaptic if they colocalized with Homer 1-DsRed dendritic clusters for at least five frames. . Immunocytochemistry Neurons were fixed in 2% paraformaldehyde, 2% sucrose and stained in non-permeabilized conditions with primary antibodies against extracellular GluN2A or GluN2B (ACG-002 and ACG-003, Alomone).

Techniques: Cell Culture, Recombinant, Software

Journal: The Journal of Neuroscience

Article Title: Vezatin Is Essential for Dendritic Spine Morphogenesis and Functional Synaptic Maturation

doi: 10.1523/JNEUROSCI.3084-11.2012

Figure Lengend Snippet: Vezatin concentrates in spines during neuronal differentiation. A, Developmental distribution of Vezatin showing its concentration in growth cone and dendrites early during neuronal differentiation (inset, left) and in dendrites and spines in maturing and mature neurons (insets, middle and right). B, At the early stage of neuronal differentiation (left) and later, at the onset of dendritic spine development, Vezatin does not colocalize with presynaptic Syb2 (right, arrow). C, Postsynaptic Vezatin is partially codistributed with transsynaptic N-cadherin. C1, The arrowhead and the arrow are pointing to a spine where Vezatin is (yellow) and is not (red aside green) clearly overlapping the N-cadherin signal, respectively. D, Vezatin is expressed in the soma of interneurons stained with GAD67 (left). Vezatin does not codistribute with GAD65 at the axonal compartment (right). MAP2 is used to label dendrite. E, Snapshot of a time-lapse image of a living cortical neuron transfected with Vezatin-GFP and RFP-actin showing codistribution in a hemicircumferential ring in a growth cone. Top, inset, DIV 3, fixed cultured hippocampal neuron immunostained for Vezatin and GluA2, a glutamate receptor, showing that Vezatin expression is restricted to an arc-shaped region of the growth cone. F, Vezatin colocalizes with phalloidin (F-actin) in dendrites and in developing spines at early synaptic sites (DIV 14). Scale bars, 10 μm. G, Protein expression in mouse hippocampi extracts. The anti-Vezatin antibody recognized the two major Vezatin isoforms (see text) with band intensities approximately 2× weaker in the heterozygous floxed/null (fl/null) compared to the floxed/floxed (fl/fl) extract. Other few alternatively spliced variants are expressed in minor quantities. Loading control, GAPDH.

Article Snippet: Rabbit antibody anti-GluA2 was used at 1:200 (Alomone Labs).

Techniques: Concentration Assay, Staining, Transfection, Cell Culture, Expressing

Journal: The Journal of Neuroscience

Article Title: Vezatin Is Essential for Dendritic Spine Morphogenesis and Functional Synaptic Maturation

doi: 10.1523/JNEUROSCI.3084-11.2012

Figure Lengend Snippet: Subcellular spine Vezatin expression in vitro (A–E) and in vivo (F–G). A, Vezatin labels all stages of spine differentiation. Insets, Higher magnifications of a filopodium (arrowhead) and a thin spine (left). A stubby (arrow) and a mushroom-like (arrowhead) are shown on the right (top and bottom insets). B, Confocal images showing that Vezatin (green) and PSD95 (red) colocalize (yellow) at spine heads. Asterisks and arrowheads point to representative mushroom and stubby spines, respectively. Bottom right, Reconstructed view using the Imaris software package. C, Vezatin extends beyond the PSD95/PSD margin toward the neck of the spine: snapshot of a confocal spine 3D reconstruction (21 DIV). Overlay (yellow) between Vezatin (green) and PSD95 (red) staining. Both markers colocalize at the tip of the spine head, but PSD95 is more central than Vezatin. D, Vezatin is in the synaptosomal membrane fraction as PSD95 or GluA2 and GluN1 (P2). Vezatin is also in the cytosolic fraction (S2). Crude protein extract was obtained by clearing the lysate by gentle centrifugation (S1) followed by high-speed centrifugation (S2 and P2). E, High-magnification images of a 21 DIV spine whose cup-shaped base is colabeled (overlay) with Vezatin (green), phalloidin (red), and PSD95 (blue). Scale bars: A, 10 μm; B (left), E, 2 μm; B (right), 1 μm. F, Vezatin is expressed in hippocampus, cortex, and MHb. Other regions including striatum [caudate putamen (CPu) and cerebral peduncle (CP)], amygdala, and thalamus [ventral postero-medial thalamic nucleus (VPM) and ventral postero-lateral thalamic nucleus (VPL)] expressed Vezatin at a low level (objective 1.6 ×). Scale bar: 1000 μm. G, Confocal microscopy at low-power resolution indicates that Vezatin is expressed in soma (objective 20×) [SP in CA1/CA3 and interior/exterior (int/ext) in DG]. No staining is observed in the hilus (DG), except in cells attributed to astrocytes and/or inhibitory neurons. DAPI staining is used to label nuclei. G1, Higher-power resolution (objective 40×, zoom 2.67). Left, Vezatin is in dendrites (SR in CA1). Middle, Vezatin is in spines partially codistributing with the transsynaptic N-cadherin marker (CA1 SR region). Inset, Arrowhead and arrow are pointing to spines where postsynaptic Vezatin (green) is (yellow) and is not (green aside red) overlapping N-cadherin (red). Right, Vezatin is in postsynaptic thorny excrescences (in SL in CA3) that mark the location of mossy fiber synaptic terminations (arrow). Scale bars: G, 400 μm; G1, 100 μm. SP, stratum pyramidale; SL, stratum lacunosum.

Article Snippet: Rabbit antibody anti-GluA2 was used at 1:200 (Alomone Labs).

Techniques: Expressing, In Vitro, In Vivo, Software, Staining, Centrifugation, Confocal Microscopy, Marker

Journal: Cell Metabolism

Article Title: A Role for p53 in the Adaptation to Glutamine Starvation through the Expression of SLC1A3

doi: 10.1016/j.cmet.2018.07.005

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Article Snippet: Rabbit monoclonal anti-AGC1 (D5I6I) , Cell Signaling Technology , Cat#64169.

Techniques: Recombinant, In Vitro, In Vivo, Cloning, Plasmid Preparation, Software, CRISPR

Journal: iScience

Article Title: GluK2 Q/R editing regulates kainate receptor signaling and long-term potentiation of AMPA receptors

doi: 10.1016/j.isci.2023.107708

Figure Lengend Snippet:

Article Snippet: Rabbit Polyclonal Anti-GluA3 , Alomone , RRID: AB_2039883.

Techniques: Recombinant, Protein Extraction, Software