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: 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

Journal: Neural Regeneration Research

Article Title: Mechanisms responsible for the effect of median nerve electrical stimulation on traumatic brain injury-induced coma: orexin-A-mediated N-methyl-D-aspartate receptor subunit NR1 upregulation

doi: 10.4103/1673-5374.184494

Figure Lengend Snippet: Effect of MNS on NR1 expression in the prefrontal cortex of rats with TBI-induced coma (western blot assay). Control group: Sham-operated rats (skin incision but no TBI). TBI group: Weight-drop method was used to establish a model of TBI. Stimulated group: Comatose rats with TBI underwent MNS. Antagonist group: Comatose rats with TBI and given MNS received an intracerebroventricular injection of the OX1R antagonist SB334867. Data are expressed as the mean ± SD ( n = 6 rats per time point per group). * P < 0.05, vs . control group; # P < 0.05, vs . TBI group; † P < 0.05, vs . stimulated group; § P < 0.05, vs . 6 h; ‡ P < 0.05, vs . 12 h (one-way analysis of variance). MNS: Electrical stimulation of the median nerve; TBI: traumatic brain injury; h: hours.

Article Snippet: The membranes were incubated with rabbit anti-NR1 polyclonal antibody (1:200; Wuhan Boster Biotechnology Co., Ltd., Wuhan, Hubei Province, China) and mouse anti-β-actin monoclonal antibody (1:500; Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) overnight at 4°C, and washed in Tris buffered saline with Tween 20.

Techniques: Expressing, Western Blot, Control, Injection

Journal: Neural Regeneration Research

Article Title: Mechanisms responsible for the effect of median nerve electrical stimulation on traumatic brain injury-induced coma: orexin-A-mediated N-methyl-D-aspartate receptor subunit NR1 upregulation

doi: 10.4103/1673-5374.184494

Figure Lengend Snippet: Effect of MNS on NR1 immunoreactivity in the prefrontal cortex of rats with TBI-induced coma (× 400). Control group: Sham-operated rats (skin incision but no TBI). TBI group: Weight-drop method was used to establish a model of TBI. Stimulated group: Comatose rats with TBI underwent MNS. Antagonist group: Comatose rats with TBI and given MNS received an intracerebroventricular injection of the OX1R antagonist SB334867. Brown cells (red arrows) are NR1-immunoreactive. Positive immunostaining for NR1 was found in the cytoplasm, cell membrane, and nucleus of neurons in the prefrontal cortex. MNS: Electrical stimulation of the median nerve; TBI: traumatic brain injury; h: hours.

Article Snippet: The membranes were incubated with rabbit anti-NR1 polyclonal antibody (1:200; Wuhan Boster Biotechnology Co., Ltd., Wuhan, Hubei Province, China) and mouse anti-β-actin monoclonal antibody (1:500; Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) overnight at 4°C, and washed in Tris buffered saline with Tween 20.

Techniques: Control, Injection, Immunostaining, Membrane

Journal: BMC Anesthesiology

Article Title: The interplay of BDNF-TrkB with NMDA receptor in propofol-induced cognition dysfunction

doi: 10.1186/s12871-018-0491-y

Figure Lengend Snippet: Immunohistochemical staining revealed that the protein levels of NMDAR and BDNF in hippocampus tissue. A. NMDAR and BDNF were expressed abundantly both in the nucleus and cytoplasm in the PND group, the improvement effect of the PNK group was inhibited compared to PN, the improvement effect of the PMD group was inhibited compared PD group. B. The mRNA expression of NMDAR, proBDNF, mBDNF, TrkB, ERK1 and CREB were tested by RT-PCR. The mRNA levels of NMDAR, mBDNF and TrkB were lower in the P group than those in the NS group, while the level of proBDNF was up-regulated in the P group compared to NS group. However, there were no obvious different expression of ERK1and CREB between the NS group and the others. C, F. The protein phosphorylation of NMDAR, proBDNF, mBDNF, TrkB, ERK1 and CREB were determined by Western blot. The phosphorylation levels of NMDAR, TrkB, ERK1 and CREB in the PND group were highest, while P group were the lowest. D, E. The ratio of p-NMDAR and NMDAR, the ratio of p-TrkB andTrkB. *versus NS group, P < 0.05; #versus P group, P < 0.05; &versus PN group, P < 0.05; $versus PD group, P < 0.05

Article Snippet: The protein expression and phosphorylation of NMDA receptor, proBDNF, mBDNF, TrkB, ERK1 and CREB were determined by Western blot using specific monoclonal antibodies as follows: rabbit anti-NMDAR (PA1222, BosterBio), rabbit anti-p-NMDAR (ABN99, Millipore), mouse anti-proBDNF (sc-65,514, Santa Cruz), rabbit anti-mBDNF (D160119, Sangon Biotech, China), rabbit anti-TrkB (ab18987, Abcam), rabbit anti-p-TrkB (ABN1381, Millipore), rabbit anti-CREB (ab32515, Abacm), rabbit anti-p-CREB (ab32096, Abcam).

Techniques: Immunohistochemical staining, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction, Phospho-proteomics, Western Blot