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: Communications Biology

Article Title: Phosphorylation of NMDA receptors by cyclin B/CDK1 modulates calcium dynamics and mitosis

doi: 10.1038/s42003-020-01393-3

Figure Lengend Snippet: a Cartoon illustrating the NMDA subunit combinations purified from cells in interphase. The cylinders represent the subunits of the NMDAR. The triangles simulate the phosphorus given by the cyclin B1/CDK1 complex (green circle/yellow square). The subunits with the alanine mutant (mA) are not phosphorylated by the complex. c , d Purified NR1wt/NR2Awt, NR1mA/NR2Awt and NR1wt/NR2AmA were incubated with a purified recombinant CyclinB1/CDK1 complex (see “Materials and methods” section) for an in vitro phosphorylation assay. b Blots using the MPM2 antibody. NR1wt blot shows in lanes 1–2 replicas of NR1wt from the combination NR1wt/NR2Awt; and lanes 3–4 replicas of NR1wt from combination NR1wt/NR2mA. NR1mA blot shows in lanes 1–4 replicas from the NR2A purified from the combination NR1mA/NR2Awt. NR2Awt blot shows in lanes 1–2 replicas of NR1wt from the combination NR1wt/NR2Awt; and lanes 3–4 replicas from the NR2A purified from the combination NR1mA/NR2Awt. Finally, NR2AmA blot shows in lanes 1–4 replicas from the NR2A purified from the combination NR1wt/NR2AmA. c Blots show in the same order the total protein content identified by Coomassie staining. d Blots show the total protein (left) and western blot using the MPM2 antibody (right) from the combination NR1wt/NR2Awt which was not incubated with the phosphorylation cocktail containing the recombinant purified Cyclin B1/CDK1 complex. Lanes 1–2 replicas from NR1wt and lanes 3–4 replicas from NR2Awt. This was used to assess the background phosphorylation of NMDAR purified from cells in interphase.

Article Snippet: NMDA receptors plasmids were acquired from Addgene: pCI-EGFP-NR1 wt (#45446) and pCI-SEP_NR2A (#23997).

Techniques: Purification, Mutagenesis, Incubation, Recombinant, In Vitro, Phospho-proteomics, Staining, Western Blot

Journal: Cells

Article Title: MC4R Localizes at Excitatory Postsynaptic and Peri-Postsynaptic Sites of Hypothalamic Neurons in Primary Culture

doi: 10.3390/cells13151235

Figure Lengend Snippet: Hypothalamic neurons in primary culture express GluN1 localized at postsynaptic sites. ( A ) An orthogonal projection of six confocal images of a primary neuron expressing endogenous GluN1 and PSD95 visualized with the rabbit anti-GluN1 (AGC-001) and anti-PSD95 K28/43 antibody, respectively. The white rectangles indicate the inset regions shown at higher magnification in ( C ), and the yellow asterisk indicates the neuron of interest. ( B ) Left and right panels: Single confocal images from different positions of a Z-stack of 6 µm thickness showing the punctate distribution of PSD95 (left) and the negative staining of the nucleus (right) in the cell soma of the neuron shown in ( A ). ( C ) Enlarged images of the inset regions in ( A ). Yellow arrowheads indicate branching points. ( D ) A representative SR image of a point of PSD95 fluorescence with a representative yellow dashed line was used to determine the average diameter of PSD95 points. The graph on the right illustrates the average +/− SEM fluorescence (gray value) of PSD95 along the line, determined by measuring 180 spots of PSD95 fluorescence from 6 neurons. ( E , F ) SR images of a neurite expressing GluN1 and PSD95. The white arrow indicates a spot of PSD95 and GluN1 colocalization, and the green/magenta arrows indicate spots where PSD95 and GluN1 are adjacent. The regions indicated by the arrows are shown enlarged in ( F ). Yellow lines indicate those drawn for line segment analyses, with corresponding graphs displayed as means +/− SEM. ( G ) The distance (nm) between GluN1 and PSD95 fluorescence peaks was determined by the line segment analyses from 6 neurons from 2 independent experiments (n = 46 colocalizing points, n = 17 adjacent points), displayed as means +/− SD. The green dotted line is placed at the 216 nm cutoff that discriminates spots at which GluN1 and PSD95 colocalize (distance of fluorescence peaks < 216 nm) and those where GluN1 and PSD95 are adjacent (distance between fluorescence peaks > 216 nm and <450 nm), as indicated by the models.

Article Snippet: Primary antibodies: rabbit anti-PSD95 (Abcam #ab18258, 1:100 dilution), mouse anti-PSD95 (K28/43, NeuroMab 75-028, 1:200 dilution), mouse anti-PSD95 (Invitrogen MA1-046 1:25 dilution), rabbit anti-GluN1 (Alomone labs #AGC-001, 2 µg/mL final concentration), rabbit anti-NR2A/GluN2A (N327/95, NeuroMab 75-288, 1:50 dilution), rabbit anti-HA (C29F4, Cell Signaling Technology # 3724, 1:100 dilution), mouse anti-HA Tag (F-7) Alexa Fluor 647 (Santa Cruz Biotechnology, sc-7392 AF647, 1:100 dilution in live cells, 1:50 dilution in fixed cells).

Techniques: Expressing, Negative Staining, Fluorescence

Journal: Cells

Article Title: MC4R Localizes at Excitatory Postsynaptic and Peri-Postsynaptic Sites of Hypothalamic Neurons in Primary Culture

doi: 10.3390/cells13151235

Figure Lengend Snippet: HA-MC4R localizes together with and in the proximity of GluN1. ( A ) Confocal images (30 µm scale bar) and SR images (5 µm scale bar) of an HA-MC4R-expressing neuron, immunostained with antibodies with mouse anti-HA Tag (F-7) and rabbit anti-GluN1 (AGC-001) to visualize endogenous GluN1. The white rectangle indicates the enlarged region. The white arrow indicates a point of colocalization, and the cyan/magenta arrows indicate a site where GluN1 and HA-MC4R are adjacent. ( B ) Enlarged images of the neurite above where HA-MC4R and GluN1 colocalize (white arrow). ( C ) Enlarged images of the neurite above where HA-MC4R and GluN1 are adjacent (magenta and cyan arrows). ( B , C ) The yellow dashed lines indicate those drawn for the segment analyses, with the corresponding graphs displayed as means +/− SEM on the right. ( D ) The distance (nm) between HA-MC4R and GluN1 fluorescence peaks was determined by line segment analyses from 3 neurons from 2 independent experiments (n = 34 colocalizing points, 21 adjacent points). The green dotted line in the graph is as in . Models indicate the range of distances between colocalizing and adjacent peaks of HA-MC4R and GluN1 fluorescence, respectively.

Article Snippet: Primary antibodies: rabbit anti-PSD95 (Abcam #ab18258, 1:100 dilution), mouse anti-PSD95 (K28/43, NeuroMab 75-028, 1:200 dilution), mouse anti-PSD95 (Invitrogen MA1-046 1:25 dilution), rabbit anti-GluN1 (Alomone labs #AGC-001, 2 µg/mL final concentration), rabbit anti-NR2A/GluN2A (N327/95, NeuroMab 75-288, 1:50 dilution), rabbit anti-HA (C29F4, Cell Signaling Technology # 3724, 1:100 dilution), mouse anti-HA Tag (F-7) Alexa Fluor 647 (Santa Cruz Biotechnology, sc-7392 AF647, 1:100 dilution in live cells, 1:50 dilution in fixed cells).

Techniques: Expressing, Fluorescence

Journal: Scientific Reports

Article Title: Functional NMDA receptors are expressed by human pulmonary artery smooth muscle cells

doi: 10.1038/s41598-021-87667-0

Figure Lengend Snippet: Functional NMDA receptors are localized on the surface of HPASMCs. Immunofluorescence staining was performed on non-permeabilized HPASMCs. Both GluN1 and GluN2 (2B and 2D) subunits were detected on the surface of HPASMCs ( A , B ). GluN1 co-localized with GluN2B ( A ) and GluN2D ( B ) on the surface of HPASMCs, respectively. Results are representative images taken from at least three separate experiments. Scale bar = 100 µM. ( C , D ) are representative scatter plots of each fluorescent pixel from confocal images with GluN1 green fluorescent intensity along the x -axis and GluN2 red fluorescent intensity along the y -axis. The shaded area in the upper right quadrant represents colocalized pixels above background with the associated Pearson correlation coefficient indicated for all colocalized pixels. ( E ) Representative traces for cultured HPASMCs in response to 100 μM NMDA and 10 μM glycine treatment. Cells were clamped at − 50 mV and whole cell recordings were performed. ( F ) NMDA and glycine treatment evoked an inward whole-cell current of 10.9 ± 1.7 pA (Mean ± SE, **p < 0.01, n = 16). ( G ) NMDA and glycine treatment evoked increased AUC compared with baseline (*p < 0.05, n = 16).

Article Snippet: After 3 washes in TBS, sections were permeabilized and pre-blocked followed by primary antibody incubation at the following concentrations: platelet endothelial cell adhesion molecule (PECAM-1) (ab28364, Abcam, Cambridge, MA) (1/150), GluN1 (556308, BD Pharmingen, San Jose, CA) (1/1000) (this reference contains characterization of the antibody against GluN1), GluN2A (AGC-002, Alomone, Jerusalem, Israel) (1/20,000), GluN2B (AGC-003, Alomone, Jerusalem, Israel) (1/3000), GluN2C (ab105146, Abcam, Cambridge, MA) (1/30,000) or GluN2D (ab186816, Abcam, Cambridge, MA) (1/10,000).

Techniques: Functional Assay, Immunofluorescence, Staining, Cell Culture