Journal: bioRxiv

Article Title: GluN2D-containing NMDA receptors regulate dentate gyrus function by facilitating granule cell activity and mediating synaptic plasticity

doi: 10.64898/2026.03.06.710109

Figure Lengend Snippet: (A) Grin2d f l/fl mice were injected with AAV5-CamKII-mCherry (Control) or AAV-CamKII-mCherry-Cre ( Grin2d cKO). NMDAR-LTP was abolished in Grin2d cKO compared with control mice (Control: 149.5 ± 6.0 %, p < 0.01, n = 5, paired t-test; cKO: 92.5 ± 5.3 %, p = 0.12201, n = 6, paired t-test; Control vs cKO: p < 0.001, unpaired t-test). (B) WT mice were bilaterally injected with an anti-GluN2D antibody or control Ab into the dentate gyrus. After one hour, animals were euthanized, and slices were prepared. Injection was confirmed by the presence of methylene blue. NMDAR-LTP was abolished in mice injected with the anti-GluN2D antibody (cKO: 110.4 ± 8.5 %, p = 0.2952, n = 6, paired t-test) compared with control mice (Control: 149.8 ± 8.1 %, p < 0.001, n = 7, paired t-test; Control vs cKO: p < 0.01, unpaired t-test). (C) NMDAR-LTP was impaired in Grid1 KO mice (KO: 117.7 ± 5.3, p < 0.05%, n = 8, Wilcoxon signed-rank test) compared with controls (Control: 147.5 ± 6.7 %, p < 0.001, n = 7, paired t-test; Control vs cKO: p < 0.05, Mann-Whitney U test). Data are presented as mean ± s.e.m.

Article Snippet: For GluN2D cross-linking experiments in C57BL/6J, the control group received 1 μL of anti-rabbit Alexa 568 (control IgG, 1/5), while the GluN2D-cross-link group received 1 μg of polyclonal rabbit anti-GluN2D subunit (Alomone Labs, cat #AGC-020), both diluted in PBS with 1% methylene blue (1 μL final volume).

Techniques: Injection, Control, MANN-WHITNEY

Journal: Frontiers in Cell and Developmental Biology

Article Title: Astrocytic gatekeeping of neural circuitry and synaptic balance in an autism mouse model: mechanistic insights beyond Gryllus bimaculatus extract-derived therapy

doi: 10.3389/fcell.2025.1677851

Figure Lengend Snippet: Protective effects of Gryllus bimaculatus (Gb) extract on abnormal expression levels of glutamatergic and GABAergic synaptic proteins in the valproic acid (VPA)-induced autism spectrum disorder (ASD) mouse brain tissues. Immunoblot analyses for GRM5, vGluT1, NMDA R1, GABA R1α, and VGAT proteins were performed on prefrontal cortex (PFC) tissue lysates collected at embryonic day 15 (E15) (A) , postnatal day 3 (P3) (B) , and P40 (C) from mice subjected to various treatment combinations. Experimental groups included CTL (saline, n = 8); VPA (600 mg/kg VPA, n = 8); VPA + Gb 5 (600 mg/kg VPA + 5 g/kg Gb extract, n = 8); VPA + Gb 10 (600 mg/kg VPA + 10 g/kg Gb extract, n = 8); Gb 5 (5 g/kg Gb extract, n = 8); Gb 10 (10 g/kg Gb extract, n = 8). Control values were normalized to 1 (mean ± SEM, n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001 compared with control; # p < 0.05, ## p < 0.01, ### p < 0.001 compared with VPA alone; ns , not significant).

Article Snippet: Antibodies against synaptic markers, including NLGN1 (#NBP2-42192), NLGN2 (#NBP2-41299), NLGN3 (#NBP2-42200), SHANK3 (#NBP1-47610), the NMDA receptor NR1 subunit (#NB300-114) from Novus Biologicals (Centennial, CO, United States) and NRXN1 (#PA5-79764) was from Thermo Fisher Scientific (Waltham, MA, United States).

Techniques: Expressing, Western Blot, Saline, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Astrocytic gatekeeping of neural circuitry and synaptic balance in an autism mouse model: mechanistic insights beyond Gryllus bimaculatus extract-derived therapy

doi: 10.3389/fcell.2025.1677851

Figure Lengend Snippet: Regulatory effects of Gryllus bimaculatus (Gb) extract on excitatory and inhibitory neuronal activity in primary cortical neurons from valproic acid (VPA)-treated embryonic mice. (A) Schematic representation of primary cortical neuron cultures derived from embryonic mouse brains. Experimental groups included CTL (saline, n = 8); VPA (600 mg/kg VPA, n = 8); VPA + Gb 5 (600 mg/kg VPA + 5 g/kg Gb extract, n = 8); VPA + Gb 10 (600 mg/kg VPA + 10 g/kg Gb extract, n = 8); Gb 5 (5 g/kg Gb extract, n = 8); Gb 10 (10 g/kg Gb extract, n = 8). (B,D) Immunoblot analyses of NMDA R1, vGluT1, GRM5, GABA R1α, VGAT, NLGN3, NRXN1, and Tuj-1 in cultured primary cortical neuron lysates. Equal amounts of protein were loaded per lane, with β-tubulin used as a loading control. The bars represent fold-changes in the densitometric values of individual protein bands relative to the corresponding β-tubulin band densities. Control values were normalized to 1 (mean ± SEM, n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001 compared with control; ## p < 0.01, ### p < 0.001 compared with VPA alone; ns , not significant). (C) Confocal microscopy images of cortical neurons from various experimental groups. Cells were cultured for 7 days, fixed, and subsequently immunostained for vGluT1 (red), with nuclei counterstained using DAPI (blue). Scale bar: 50 μm.

Article Snippet: Antibodies against synaptic markers, including NLGN1 (#NBP2-42192), NLGN2 (#NBP2-41299), NLGN3 (#NBP2-42200), SHANK3 (#NBP1-47610), the NMDA receptor NR1 subunit (#NB300-114) from Novus Biologicals (Centennial, CO, United States) and NRXN1 (#PA5-79764) was from Thermo Fisher Scientific (Waltham, MA, United States).

Techniques: Activity Assay, Derivative Assay, Saline, Western Blot, Cell Culture, Control, Confocal Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: Astrocytic gatekeeping of neural circuitry and synaptic balance in an autism mouse model: mechanistic insights beyond Gryllus bimaculatus extract-derived therapy

doi: 10.3389/fcell.2025.1677851

Figure Lengend Snippet: Crucial role of astrocytes in excitatory and inhibitory (E/I) neurotransporter activities in Gryllus bimaculatus (Gb) extract-treated mixed cultures from valproic acid (VPA)-treated mouse brain. (A) Schematic representation of three different types of mixed culture systems derived from embryonic and postnatal mouse brains: Type 1, astrocytes from each treatment group combined with neurons from untreated mice; Type 2, astrocytes from untreated mice combined with neurons from each treatment group; Type 3, astrocytes and neurons both derived from the same treatment group. Astrocytes from postnatal day 3 mouse brains were seeded for 7 days, followed by the addition of cortical neurons from embryonic day 15 mouse brains onto astrocytes monolayers for an additional 7 days. (B–D) Confocal microscopy images of the different types of mixed cultures. Cells were fixed and immunostained for Tuj-1 (green) and GFAP (purple), with nuclei counterstained using DAPI (blue). Scale bar: 50 μm. Experimental groups included CTL (saline, n = 8); VPA (600 mg/kg VPA, n = 8); VPA + Gb 5 (600 mg/kg VPA + 5 g/kg Gb extract, n = 8); VPA + Gb 10 (600 mg/kg VPA + 10 g/kg Gb extract, n = 8); Gb 5 (5 g/kg Gb extract, n = 8); Gb 10 (10 g/kg Gb extract, n = 8). (E) Western blots analysis of type III mixed culture. Cell lysates were immunoblotted for Tuj-1, GFAP, synaptophysin, NMDA receptor 1 (NMDA R1), GABA receptor 1α (GABA R1α), EAAT1, and EAAT2. Equal amounts of protein were loaded per each lane, with β-actin serving as the loading control. Bars represent fold-changes in the densitometric values of the bands relative to the corresponding β-actin densities. Control values were normalized to 1 (mean ± SEM, n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001 compared with control; # p < 0.05, ## p < 0.01, ### p < 0.001 compared with VPA alone; ns , not significant).

Article Snippet: Antibodies against synaptic markers, including NLGN1 (#NBP2-42192), NLGN2 (#NBP2-41299), NLGN3 (#NBP2-42200), SHANK3 (#NBP1-47610), the NMDA receptor NR1 subunit (#NB300-114) from Novus Biologicals (Centennial, CO, United States) and NRXN1 (#PA5-79764) was from Thermo Fisher Scientific (Waltham, MA, United States).

Techniques: Derivative Assay, Confocal Microscopy, Saline, Western Blot, Control

Journal: Journal of Neurochemistry

Article Title: Pharmacological target sites for restoration of age‐associated deficits in NMDA receptor‐mediated norepinephrine release in brain

doi: 10.1111/jnc.16280

Figure Lengend Snippet: Characterizing the cellular localization of the NMDA receptors regulating [ 3 H] ‐NE release in young rat cortical brain slices. 1 mM Glu‐stimulated‐[ 3 H]‐NE releases in the cerebral cortex tissue slices from young rats ( n = 3–7) in the presence 1 & 3 μM of the TTX, voltage‐gated Na channel blocker, 10 μM MK‐801, and a combination of MK‐801 and TTX. Data are expressed as mean (±SEM) of net fractional release (stimulated—basal), with each data point representing a duplicate from one animal. Data were analyzed using a mixed‐effect analysis followed by Dunnett's multiple comparison test **** p < 0.0001. NE, norepinephrine; Glu, glutamate; TTX, tetrodotoxin.

Article Snippet: The NMDA receptor NR2A and NR2B subunit polyclonal antibodies were used alongside the NR1 monoclonal antibody (PhosphoSolutions, CO, USA; Cat # 1805‐NR1).

Techniques: Comparison

Journal: Journal of Neurochemistry

Article Title: Pharmacological target sites for restoration of age‐associated deficits in NMDA receptor‐mediated norepinephrine release in brain

doi: 10.1111/jnc.16280

Figure Lengend Snippet: The stimulatory effect of Glutamate Vs. NMDA on [ 3 H] ‐NE release in young rat cortical brain slices. In (a), the concentrations‐response curves of glutamate and NMDA‐stimulated [ 3 H] ‐NE releases in the cerebral cortex tissue slices from young rats ( n = 4). In (b), the 1 mM glutamate and NMDA stimulated NE release in the presence and absence of 1.2 mM magnesium in the cerebral cortex tissue slices from young rats ( n = 3). Data were analyzed using an unpaired t ‐test. * p ≤ 0.05, ** p ≤ 0.01 NE, norepinephrine; Glu, glutamate; NMDA, N‐methyl‐ d ‐aspartate; Mg 2+ , magnesium.

Article Snippet: The NMDA receptor NR2A and NR2B subunit polyclonal antibodies were used alongside the NR1 monoclonal antibody (PhosphoSolutions, CO, USA; Cat # 1805‐NR1).

Techniques:

Journal: Journal of Neurochemistry

Article Title: Pharmacological target sites for restoration of age‐associated deficits in NMDA receptor‐mediated norepinephrine release in brain

doi: 10.1111/jnc.16280

Figure Lengend Snippet: Age‐associated changes in the expression of NMDA receptors freely solubilize subunits in the cortical rat tissue homogenate. A representative Western blot for NMDA receptors subunits in the cerebral cortex in (a); and in (b) quantified results for GluN1, GluN2A, and GluN2B expressions in young and aged rats ( n = 5). Data are expressed as mean (±SEM) normalized to young rats, with each data point representing a duplicate from one animal. Data were analyzed using an unpaired t ‐test. * p ≤ 0.05. NMDA, N‐methyl‐ d ‐aspartate).

Article Snippet: The NMDA receptor NR2A and NR2B subunit polyclonal antibodies were used alongside the NR1 monoclonal antibody (PhosphoSolutions, CO, USA; Cat # 1805‐NR1).

Techniques: Expressing, Western Blot

Journal: Journal of Neurochemistry

Article Title: Pharmacological target sites for restoration of age‐associated deficits in NMDA receptor‐mediated norepinephrine release in brain

doi: 10.1111/jnc.16280

Figure Lengend Snippet: Effect of aging on [ 3 H]‐MK‐801 binding to NMDA receptors in the young (2–3 months old) and aged (18–24 months old) rat cortical tissue membrane. In (a) saturation curve of [ 3 H]‐MK‐801 binding to NMDA receptors in young rats in the presence of 10 μM Glu and Gly ( n = 4). The non‐linear least‐squares fitting of the saturation isotherm yielded K d and B max values of 1.8 nM and 970 fmol/mg of protein, respectively. Both total and non‐specific binding of [ 3 H]‐MK‐801 is shown in the curve, and the specific [ 3 H]‐MK‐801 binding is presented with 95% CI in dotted lines. Inset: Saturation data graphed as Scatchard plots. Whereas in (b), the binding of 10 nM [ 3 H]‐MK‐801 in +/− 10 μM Glu and Gly in young and aged rats ( n = 7), each performed in triplicate and repeated twice. In (c), the % increases after subtracting baseline binding from the binding in the presence of 10 μM Glu and Gly. Baseline Binding values represent [ 3 H]‐MK‐801 binding without exogenous addition of Glu and Gly. Data were analyzed using a Mixed‐effect analysis followed by Tukey's multiple comparison tests. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001; whereas the % of increase over the baseline was analyzed using an unpaired t ‐test: ** p ≤ 0.01. NMDA, N‐methyl‐d‐aspartate; Glu, Glutamate; Gly, Glycine; K d , dissociation constant; B max , Maximum Binding; n H , Hill Coefficient).

Article Snippet: The NMDA receptor NR2A and NR2B subunit polyclonal antibodies were used alongside the NR1 monoclonal antibody (PhosphoSolutions, CO, USA; Cat # 1805‐NR1).

Techniques: Binding Assay, Membrane, Comparison

Journal: Journal of Affective Disorders Reports

Article Title: Long-term cyclosporine A treatment promotes anxiety-like behavior: Possible relation with glutamate signaling in rat hippocampus

doi: 10.1016/j.jadr.2022.100394

Figure Lengend Snippet: Fig. 4. Western blot analysis of NMDA receptors in the hippocampus of control and CsA-treated rats. (A) Representative images of the levels of total (pan) GluN2, GluN2A, GluN2B, p-GluN1 (Ser897) and total GluN1. (B-F) Respective quantifications of immunodetections normalized to β-actin and presented as a percentage of the control mean. Data represent the mean ± SEM. Statistics: ** *p ≤0.001 as determined by Student’s t-test; n = 6.

Article Snippet: The utilized primary antibodies and respective dilutions were as follows: anti-AMPAR (1:500, #13185 Cell Signaling Technology), phosphorylated AMPAR (pAMPAR-S831) (1:1000, A4352-Sigma–Aldrich), pAMPAR (S845) (1:1000, #8084 – Cell Signaling Technology), GluN1 (1:750, #G8913 – Sigma–Aldrich), pan-GluN2 (1:750, 244-0P – SYSY Synaptic Systems), GluN2A (1:1000, #4205 - Cell Signaling Technology), GluN2B (1:1000, #4207 - Cell Signaling Technology), pGluN1 (Ser897) (1:500, 3385S – Cell Signaling Technology), nNOS (1:500, SC648 – Santa Cruz Biotechnology), and iNOS (1:750, SC7271 – Santa Cruz Biotechnology). β-actin antibody (1:5000, A5441 – Sigma-Aldrich) was used as a loading control in the experiments.

Techniques: Western Blot, Control