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: The Journal of physiology

Article Title: Neurones in the supraoptic nucleus of the rat are regulated by a projection from the suprachiasmatic nucleus.

doi: 10.1111/j.1469-7793.1997.149bl.x

Figure Lengend Snippet: Figure 3. PSTHs to illustrate the effects of excitatory and inhibitory antagonists on the SCN- evoked responses of three continuously firing SON cells in vitro A, the non-NMDA antagonist CNQX (10 ìÒ) reduced the excitatory effects of SCN stimulation. The NMDA antagonist APV (10 ìÒ) did not influence the effects of SCN stimulation on this cell (B), but reduced the excitatory effects of SCN stimulation on three other cells (C). D, in another cell, bicuculline (20 ìÒ) blocked the inhibitory effects of SCN stimulation. (Bin width, 5 ms for A and B, 10 ms for C and 20 ms for D; stimulus at time 0; 300 sweeps.)

Article Snippet: The following drugs were used to antagonize different neurotransmitter agents: bicuculline methiodide (Sigma) to antagonize GABAA receptors; a¬_2-amino-5-phosphonovaleric acid (APV; Sigma) to antagonize NMDA glutamate receptors; and 6-cyano-7nitroquinoxaline-2,3-dione (CNQX; Tocris Cookson) and 6-nitro-7sulphamoylbenzo(f)quinoxaline-2,3-dione (NBQX; Tocris Cookson) to antagonize non-NMDA glutamate receptors.

Techniques: In Vitro

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: bioRxiv

Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

doi: 10.1101/2020.06.03.130559

Figure Lengend Snippet: (A-E) Postnatal development of callosal projection in S1. (A) EGFP plasmid injected into lateral ventricle of embryo at embryonic day15.5 (E15.5) and electrical pulse given to enable the plasmid to enter cortical progenitor cells of layer II/III in the ventricular zone. (B, B’) At postnatal day 5 (P5), the callosal axons from S1 had reached the white matter underneath contralateral S1. (C, C’) At P8, the callosal axons were diffusely distributed in contralateral S1. (D, D’) By P12, pruning of excess projections led to a refined innervation pattern with a narrow band limited to the S1/S2 border. (E, E’) After P12, the pattern was stable as observed at P30. (F) In P14 control mice ( Emx1 cre/+ ; NR1 fl/wt ), the callosal innervation pattern of S1 of the contralateral cortex is well-differentiated with a dense innervation at S1/S2 border. The pattern persists to P30 (J). (G) In NR1 KO mice ( Emx1 cre/+ ; NR1 fl/fl ), the innervation pattern was disrupted and projections were extremely diffuse which also persisted to P30 (K). (H) Quantification of fluorescent intensity across the medial to lateral extent of the S1. (I, L) Quantification of fluorescence density of S1 region of control vs. NR1 KO mice at P14 (I, P = 0.002) and P30 (L, P = 0.0003) Scale bar: 500μm for all images. S1: primary somatosensory cortex; S2: secondary somatosensory cortex.

Article Snippet: Antibodies for intraventricular injection : commercial anti-NMDAR antibody is against amino acid residues 385-399 in the N-terminus of NR1 and was made in Rabbit (AGC-001, Alomone labs).

Techniques: Plasmid Preparation, Injection, Control, Fluorescence

Journal: bioRxiv

Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

doi: 10.1101/2020.06.03.130559

Figure Lengend Snippet: Examples of 12-μm coronal brain sections from P8 Emx1 cre/+ ; NR1 wt/wt (A) and Emx1 cre/+ ; NR1 fl/fl (B) of the same litter. Immunostaining of vesicular glutamate transporter 2 (VGult2) showed thalamocortical barrels in Layer IV of S1 which are pointed out by arrows. The VGlut2 staining in Emx1 cre/+ ; NR1 wt/wt mice revealed a clear barrel pattern (Aa). However, the barrel pattern in Emx1 cre/+ ; NR1 fl/fl mice was disrupted and less distinct (Ba). The NR1 staining in Emx1 cre/+ ; NR1 wt/wt mice were dense and strong in cortex (Ab, Ac). However, the staining in Emx1 cre/+ ; NR1 fl/fl mice was less bright and apparently reduced in Layer V and VI (Bb, Bc). Scale bar: 100μm for Ac and Bc; 500μm for rest of images.

Article Snippet: Antibodies for intraventricular injection : commercial anti-NMDAR antibody is against amino acid residues 385-399 in the N-terminus of NR1 and was made in Rabbit (AGC-001, Alomone labs).

Techniques: Immunostaining, Staining

Journal: bioRxiv

Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

doi: 10.1101/2020.06.03.130559

Figure Lengend Snippet: (A, B) The callosal axons in S1 formed a bundle and grew into the ipsilateral CC at P0 in control and NR1 KO littermates ( Emx1 cre/+ ; NR1 fl/wt and Emx1 cre/+ ; NR1 fl/fl mice). The arrows show the extent of axon growth into the CC. By P3, the callosal axons crossed the midline (C, D) and by P5, the callosal axons have grown to underneath the contralateral S1 (E, F). Scale bar: 500μm for all images.

Article Snippet: Antibodies for intraventricular injection : commercial anti-NMDAR antibody is against amino acid residues 385-399 in the N-terminus of NR1 and was made in Rabbit (AGC-001, Alomone labs).

Techniques: Control

Journal: bioRxiv

Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

doi: 10.1101/2020.06.03.130559

Figure Lengend Snippet: (A, A’) At P6, most axons in control grew into deeper layer VI of S1 (see “*”); a few axons grew to layer V from medial to lateral S1 (see arrows). However, axons projecting to lateral S2 had grown to layer IV which was apparently faster than the axons in S1 (see arrows). (B, B’) In NR1 KO mice, most axons had grown to layer V and some even grew to layer I (see arrows) at P6. (C, D) At P8, axons in control and mutant mice had grown to the superficial layer of cortex. However, the innervation patterns were different. Controls showed more axon innervation in the lateral S1 with dense callosal innervation at S1/S2 border (C). Mutants showed slightly more axon innervation in the medial S1 (D). (E) The fluorescence density of mutant mice in S1 was significantly higher than in control mice at P6 which suggested that the mutants had increased axon innervation in contralateral S1 at P6. P = 0.003. Scale bar: 500μm for all images. The square brackets in all images outline the S1. The arrow heads in all images outline the S1/S2 border. White lines outline different layers in the cortex of Figure A-D. M: medial; L: lateral.

Article Snippet: Antibodies for intraventricular injection : commercial anti-NMDAR antibody is against amino acid residues 385-399 in the N-terminus of NR1 and was made in Rabbit (AGC-001, Alomone labs).

Techniques: Control, Mutagenesis, Fluorescence

Journal: bioRxiv

Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

doi: 10.1101/2020.06.03.130559

Figure Lengend Snippet: (A) In control mice ( Emx1 cre/+ ; NR1 fl/wt ), cleaved caspase-3 + cells were mostly detected in layer II/III of M1 (A’), only rare cell death was observed in other cortical regions, such as S1 (A’’). (B) Compared with controls, there was increased cell death in layer II/III of motor cortex in mutant mice ( Emx1 cre/+ ; NR1 fl/fl ) (B’). However, compared with controls, there was no increased cell death in other cortical regions in mutant mice, such as S1 (B’’). Scale bar: 500μm for A and B; 200μm for A’, A’’, B’ and B’’.

Article Snippet: Antibodies for intraventricular injection : commercial anti-NMDAR antibody is against amino acid residues 385-399 in the N-terminus of NR1 and was made in Rabbit (AGC-001, Alomone labs).

Techniques: Control, Mutagenesis

Journal: bioRxiv

Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

doi: 10.1101/2020.06.03.130559

Figure Lengend Snippet: (A-D) Deleting NMDAR specifically in projecting neurons. Vectors expressing Cre-recombinase (Cre) and EGFP were delivered into S1 of floxed NR1 mice ( NR1 fl/wt x NR1 fl/wt ) by in utero electroporation at E15.5 (A). Callosal innervation patterns at P14 in control NR1 ipsiS1+/+ mice (B) and NR1 ipsiS1−/− mice (C). (D) Quantification of fluorescence density. P = 0.317. (E-H) Deleting NMDAR specifically in target neurons. NR1 was deleted in target contralateral S1 by in utero electroporation of Cre at E12.5 in NR1 fl/fl ; Ai14 fl/fl mice, the ipsilateral projecting neurons were labeled by EGFP at E15.5 (E). Compared with control NR1 wt/wt ; Ai14 fl/fl (F), NR1 fl/fl ; Ai14 fl/fl mice which specifically deleted NR1 in target S1 showed increased callosal innervation in S1 as “*” shows (G). (H) Quantification of fluorescence density. P = 0.002. Scale bar: 500μm for all images.

Article Snippet: Antibodies for intraventricular injection : commercial anti-NMDAR antibody is against amino acid residues 385-399 in the N-terminus of NR1 and was made in Rabbit (AGC-001, Alomone labs).

Techniques: Expressing, In Utero, Electroporation, Control, Fluorescence, Labeling

Journal: bioRxiv

Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

doi: 10.1101/2020.06.03.130559

Figure Lengend Snippet: (A) NR1 was deleted in target contralateral S1 by in utero electroporation of Cre at E13.5 in NR1 fl/fl ; Ai14 fl/fl mice, the ipsilateral projecting neurons were labeled by EGFP at E15.5. Compared with control NR1 wt/wt ; Ai14 fl/fl (B), NR1 fl/fl ; Ai14 fl/fl , with NR1 specifically deleted in upper cortical layers did not show increased callosal innervation in (C). (D) Quantification of fluorescence density. P = 0.27. Scale bar: 500μm for all images.

Article Snippet: Antibodies for intraventricular injection : commercial anti-NMDAR antibody is against amino acid residues 385-399 in the N-terminus of NR1 and was made in Rabbit (AGC-001, Alomone labs).

Techniques: In Utero, Electroporation, Labeling, Control, Fluorescence

Journal: bioRxiv

Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

doi: 10.1101/2020.06.03.130559

Figure Lengend Snippet: (A) Anti-NR1 antibodies were injected into the lateral ventricle from P2 to P8 and mice were perfused 3 hours later after last injection. Rabbit IgG served as control. Mouse brains then were stained with anti-Rabbit secondary coupled to Alexa594. The red fluorophore of Alexa594 indicated where the antibodies had distributed to. Scale bar: 500μm for all images. (B, B’) In control, the fluorescence signals were mostly detected in the cortex of the ipsilateral injection side, and few in the contralateral cortex. In the ipsilateral injection side, the signals were detected in all the cortical layers, but most strongly in the pia, layer I, layer V, layer VI, cingulum and corpus callosum (see arrows). The signals were also detected in the hippocampus and contralateral motor cortex (see arrows). (C, C’) The general antibody distribution pattern was similar as seen in control. Moreover, the anti-NR1 antibody can bind to NMDAR on the cell membranes, which thus showing beautiful cell membrane staining (see arrows in C’). Scale bar: 500 μm for Figure B, C; 200 μm for Figure B’, C’. CC: corpus callosum; cg: cingulum; Hip: hippocampus; M: motor cortex; S1: primary somatosensory cortex; S2: secondary somatosensory cortex.

Article Snippet: Antibodies for intraventricular injection : commercial anti-NMDAR antibody is against amino acid residues 385-399 in the N-terminus of NR1 and was made in Rabbit (AGC-001, Alomone labs).

Techniques: Injection, Control, Staining, Fluorescence, Membrane

Journal: bioRxiv

Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

doi: 10.1101/2020.06.03.130559

Figure Lengend Snippet: (A-D) Anti-NR1 antibodies were injected into the lateral ventricle from P2 to P12 in ipsilateral cortex. RbIgG served as control. Compared with control (B), antibody injection in mice did not show increased callosal innervation in S1 at P14 (C). (D) Quantification of fluorescence density. P = 0.94. (E-H) Anti-NR1 antibodies were injected into the lateral ventricle from P2 to P12 in contralateral cortex. Compared with control (F), antibody injection in mice showed increased callosal innervation in S1 at P14 (see “*”, G). (H) Quantification of fluorescence density. P =0.0002. Scale bar: 500μm for all images.

Article Snippet: Antibodies for intraventricular injection : commercial anti-NMDAR antibody is against amino acid residues 385-399 in the N-terminus of NR1 and was made in Rabbit (AGC-001, Alomone labs).

Techniques: Injection, Control, Fluorescence

Journal: bioRxiv

Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

doi: 10.1101/2020.06.03.130559

Figure Lengend Snippet: (A-D) Anti-NR1 antibodies were injected into the lateral ventricle from P4 to P8 in contralateral cortex. RbIgG served as control. Compared with control (B), antibody injection in mice show increased callosal innervation in S1 at P14 (C). (D) Quantification of fluorescence density. P = 0.004. (E-H) Anti-NR1 antibodies were injected into the lateral ventricle from P8 to P14 in contralateral cortex. Compared with control (F), antibody injection in mice did not show increased callosal innervation in S1 at P14 (G). (H) Quantification of fluorescence density. P = 0.69. Scale bar: 500μm for all images.

Article Snippet: Antibodies for intraventricular injection : commercial anti-NMDAR antibody is against amino acid residues 385-399 in the N-terminus of NR1 and was made in Rabbit (AGC-001, Alomone labs).

Techniques: Injection, Control, Fluorescence

Journal: bioRxiv

Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

doi: 10.1101/2020.06.03.130559

Figure Lengend Snippet: (A) The callosal innervation pattern in S1 at P30 in control mice ( Emx1 cre/+ ; NR2A fl/wt ) is similar as the pattern in P14 WT control mice, with few axons in S1 but a dense innervation at S1/S2 border. (B) In the mutant mice ( Emx1 cre/+ ; NR2A fl/fl ), the general innervation pattern was as same as control. However, the increased callosal innervation at the border of M1 and S1 was persistent at P30 (see “*” in B’). (C) Quantification of fluorescence density. P = 0.63. (D) In control Emx1 cre/+ ; NR2B fl/wt mice, the callosal innervation pattern at P30 was as normal as WT control. (E) However, the increased callosal innervation in Emx1 cre/+ ; NR2B fl/fl mice lasted at least to P30 as we observed in Emx1 cre/+ ; NR1 fl/fl mice at P30. (F) Quantification of fluorescence density. P = 0.007. Scale bar: 500μm for all images.

Article Snippet: Antibodies for intraventricular injection : commercial anti-NMDAR antibody is against amino acid residues 385-399 in the N-terminus of NR1 and was made in Rabbit (AGC-001, Alomone labs).

Techniques: Control, Mutagenesis, Fluorescence

Journal: bioRxiv

Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

doi: 10.1101/2020.06.03.130559

Figure Lengend Snippet: (A, B) EPHB2 protein expression are decreased in Emx1 cre/+ ; NR1 fl/fl mice at P5. In control Emx1 cre/+ ; NR1 wt/wt mice, EPHB2 was expressed both in CC and cortex (A). EPHB2 in Emx1 cre/+ ; NR1 fl/fl mice was dramatically decreased in cortex (B). (C) Western blot analysis of cortical protein extracts from P8 S1 showed that, relative to the loading control beta-tubulin (β-Tub) and GAPDH, lower levels of EPHB2 were observed in the five samples of Emx1 cre/+ ; NR1 fl/fl mice compared to the five samples of controls. (D) Quantification of protein levels relative to β-Tub. P=0.001. (E) Quantification of protein levels relative to GAPDH. P< 0.0001. (F) The quantitative polymerase chain reaction (qPCR) analysis showed no expression difference of EPHB2 between Emx1 cre/+ ; NR1 fl/fl mice and controls. Scale bar: 500μm for A, B.

Article Snippet: Antibodies for intraventricular injection : commercial anti-NMDAR antibody is against amino acid residues 385-399 in the N-terminus of NR1 and was made in Rabbit (AGC-001, Alomone labs).

Techniques: Expressing, Control, Western Blot, Real-time Polymerase Chain Reaction

Journal: Annals of neurology

Article Title: Absence of Neuronal Autoantibodies in Neuropsychiatric Systemic Lupus Erythematosus.

doi: 10.1002/ana.25908

Figure Lengend Snippet: Figure 1. Clinical features and immunoglobulin G (IgG) binding to conformational neuronal surface epitopes from plasma and cerebrospinal fluid (CSF) of patients with neuropsychiatric lupus (NPSLE). A. Clinical features of 35 patients with SLE, including 15 with active NPSLE at the time of sampling. 32/34 (94%) were on immunotherapies, including prednisolone (71%), hydroxychloroquine (41%), belimumab (6%) and rituximab (3%). ANA = anti-nuclear antibody. B. Live cell-based assays employing HEK293T cells with surface expressed N-methyl D-aspartate receptors (NMDARs) as NR1-NR2A or NR1-NR2B heteromers (nuclei highlighted with DAPI, 4′,6-diamidino-2-phenylindole). First panel shows that a commercial antibody directed against the extracellular domain of the NR2A-subunit (red) binds to HEK293T cells which express NR1-NR2A heteromers. NMDAR-antibody encephalitis (NMDAR-Ab-E) patient serum IgGs (n=11) bind to the NR1-NR2A heteromers (second panel), and to NR1-NR2B heteromers / NR1 homomers (data not shown). NMDAR-antibody patient CSFs show similar binding (as Irani et al, 2010).10 Serum from healthy controls (n=36), plasma and CSFs from SLE patients (n=35 of each) show no binding. No binding was observed with the G11 antibody (data not shown, lower panel representative). Throughout, IgG binding was visualized with an anti-human 568-Alexaflour antibody (1:750 dilution, A-21090) C. Commercial antibodies against the extracellular domains of NR1 (Alomone, AGC-001), NR2A (Alomone,

Article Snippet: Throughout, IgG binding was visualized with an anti-human 568-Alexaflour antibody (1:750 dilution, A-21090) C. Commercial antibodies against the extracellular domains of NR1 (Alomone, AGC-001), NR2A (Alomone, AGC-002) and NR2B (kind gift from Prof Stephenson, London) subunits bound to the surface of live HEK293T cells co-transfected with EGFP plus NR1 and NR2B subunits.

Techniques: Binding Assay, Clinical Proteomics, Sampling

Journal: Frontiers in Neuroscience

Article Title: Altered Light Sensitivity of Circadian Clock in Shank3 +/– Mouse

doi: 10.3389/fnins.2021.604165

Figure Lengend Snippet: Immunostaining for cholera toxin β-subunit (CTB) and NMDAR2A in the suprachiasmatic nucleus (SCN) from wild-type (WT) and Shank3 +/– mice. CTB (diaminobenzidine, DAB, left column ) and NMDAR2A ( right column ) did not show obvious differences in the immunoreactivity between groups (WT, top micrographs ; Shank3 +/– , bottom micrographs ). For this and subsequent figures ( , ), scale bar = 50 μm.

Article Snippet: Primary antibodies were diluted as indicated in 0.1 M PBS containing 1.0% normal serum in 0.3% Triton X-100 [anti-VIP raised in rabbit, CAT 20077, Incstar, 1:2,000 ( ); anti-CTB subunit raised in goat, CAT 703, List Biological Laboratories, 1:2,000 ( ); anti-NMDAR2A (GluN2A) raised in rabbit, CAT ACG 002, Alomone Labs, 1:400 ( ); and anti c-FOS raised in rabbit, CAT SC-52, Santa Cruz, 1:1,000 ( )].

Techniques: Immunostaining

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