Journal: Scientific Reports

Article Title: Hyaluronic acid based extracellular matrix regulates surface expression of GluN2B containing NMDA receptors

doi: 10.1038/s41598-017-07003-3

Figure Lengend Snippet: ECM removal enhances GluN2B-NMDAR mediated synaptic currents. ( A ) Example traces of NMDAR - mediated sEPCSs before and after Hya treatment in dissociated hippocampal cultures DIV21-24. ( B ) Amplitudes of single peaks show no significant differences between Hya treated or Hya plus Ifenprodil treated cultures (Ctl, −905.5 ± 179.4, n = 10; Hya, −776.2 ± 174.8, n = 10; Hya + Ifen, −758.2 ± 161.7, n = 11; average ± SEM; One-way ANOVA, P = 0.7991). ( C ) Average of single peaks before and after Hya treatment and after Ifenprodil application. Normalization of the amplitude illustrates the increased decay-time after Hya treatment (red line) in comparison to Ctl (black line). This can be restored after Ifenprodil application (green line). Ctl traces are identical. ( D ) Quantification of the area under the curve (AUC) of averaged and normalized events (left), which represent the total charge transfer revealed bigger charge transfer after ECM removal, which was reduced to control levels after blocking GluN2B-NMDAR with Ifen (Ctl, 1 ± 0.02, n = 10; Hya, 1.38 ± 0.09, n = 10; Hya + Ifenprodil, 0.98 ± 0.05, n = 11; average ± SEM; One-way ANOVA, P < 0.0001, Dunnett’s Multiple Comparison Test, ***P < 0.05).

Article Snippet: The following commercial antibodies were used for Immunocytochemistry (ICC) and Western blot (WB) in the concentrations indicated: rabbit (rb) antibodies against GluN2B (alomone labs; ICC live staining: 1:200, fixed staining.

Techniques: Blocking Assay

Journal: Scientific Reports

Article Title: Hyaluronic acid based extracellular matrix regulates surface expression of GluN2B containing NMDA receptors

doi: 10.1038/s41598-017-07003-3

Figure Lengend Snippet: ECM removal leads to increased surface expression of GluN2B in a β1 - integrin dependent manner. ( A ) Dissociated hippocampal cultures were treated with Hya over night and stained against the total amount of GluN2B and the dendritic marker Map2 (scale bar: 10 μm. ( B ) Total GluN2B expression is not affected by ECM removal (Dendrites: Ctl 1 ± 0.10, n = 30; Hya 0.89 ± 0.03, n = 30, P = 0.31; Synapses: Ctl: 1 ± 0.03, n = 30; Hya: 1.05 ± 0.03, n = 30, P = 0.27; average ± SEM; unpaired t-test). ( C ) Quantitative WB of lysed cortical cultures (DIV21) pretreated with Hya over night show no significant change in GluN2B immunoreactivity. ( D ) Dissociated hippocampal cultures at DIV21-24 were treated with Hya over night and stained against surface GluN2B (green) and the synaptic marker PSD-95 (scale bar: 10 μm). ( E ) Synaptic GluN2B surface expression at various time points after Hya treatment (Ctl: 1 ± 0.04, n = 24; Hya 1,5 h: 1.08 ± 0.04, n = 22, P = 0.76; Hya 3 h: 1.40 ± 0.09, n = 30, P = 0.0001; Hya 6 h: 1.41 ± 0.13, n = 9, P = 0.002; Hya 12 h: 1.35 ± 0.08, n = 8, P = 0.01; Hya 48 h: 1.18 ± 0.05, n = 8, P = 0.04 average ± SEM; One way-ANOVA, Dunnett’s Multiple Comparison Test). ( F,G ) GluN2B surface expression at synapses and dendrites increases after ECM degradation and can be restored by simultaneous application of the β1-integrin function blocking antibody CD29. ( F ) Synapses: Ctl: 1.0 ± 0.05, n = 68; Hya: 1.3 ± 0.05, n = 70; Hya + CD29: 0.93 ± 0.03, n = 51. ( G ) Dendrites: Ctl 1.00 ± 0.04, n = 36; Hya 1.78 ± 0.11, n = 35; Hya + CD29 0.96 ± 0.03, n = 34; average ± SEM; One-way ANOVA, P < 0.0001, Dunnett’s Multiple Comparison Test, ***P < 0.001). No ECM dependent regulation in hippocampal cultures at DIV11 (Synpases: Ctl: 1.00 ± 0.04, n = 25, Hya: 0.98 ± 0.03, n = 24, average ± SEM, unpaired t-test, P = 0.7341; Dendrites: Ctl: 1,000 ± 0.03, n = 39, Hya: 0.99 ± 0.05, n = 40; average ± SEM, unpaired t-test, P = 0.9488).

Article Snippet: The following commercial antibodies were used for Immunocytochemistry (ICC) and Western blot (WB) in the concentrations indicated: rabbit (rb) antibodies against GluN2B (alomone labs; ICC live staining: 1:200, fixed staining.

Techniques: Expressing, Staining, Marker, Blocking Assay

Journal: Scientific Reports

Article Title: Hyaluronic acid based extracellular matrix regulates surface expression of GluN2B containing NMDA receptors

doi: 10.1038/s41598-017-07003-3

Figure Lengend Snippet: ECM digestion increases p1472-GluN2B level and decreases the endocytosis of GluN2B. ( A )Dissociated hippocampal cultures at DIV21-24 were treated with Hya over night and endocytosed GluN2B (green) was quantified using Map2 staining as mask (red). ( B ) There is less endocytosis of GluN2B after ECM removal within 30 minutes (Ctl 1.00 ± 0.02, n = 79; Hya 0.9 ± 0.02, n = 80; average ± SEM, Unpaired t-test, **P = 0.0015. Scale bar: 5 µm). ( C ) Quantitative WB from lysates of acute hippocampal slices treated with Ctl or Hya probed with an antibody against pGluN2B pTyr1472 (AP2 binding site) and GluN2B. ( D ) Quantification of WB of acute hippocampal slices and cortical cultures (DIV 21–24) revealed that the amount of phosphorylated GluN2B, normalized to the total amount of GluN2B, is increased after Hya treatment (overnight for cultures, 3 h for slices; slices: Ctl 1.00 ± 0.06, n = 4; Hya 1.23 ± 0.09, n = 4; cultures: Ctl 1.00 ± 0.05, n = 9; Hya 1.26 ± 0.1, n = 9; Unpaired t-test, cultures: P = 0.0332, slices P = 0.0837, ***P < 0.0001).

Article Snippet: The following commercial antibodies were used for Immunocytochemistry (ICC) and Western blot (WB) in the concentrations indicated: rabbit (rb) antibodies against GluN2B (alomone labs; ICC live staining: 1:200, fixed staining.

Techniques: Staining, Binding Assay

Journal: Frontiers in Neuroanatomy

Article Title: Contrasting patterns of extrasynaptic NMDAR-GluN2B expression in macaque subgenual cingulate and dorsolateral prefrontal cortices

doi: 10.3389/fnana.2025.1553056

Figure Lengend Snippet: Post-embedding immunoEM for NMDA-GluN2B reveals labeling in the postsynaptic density of layer III rhesus macaque dlPFC spines. Electron micrograph depicting a layer III dlPFC spine receiving a synapse (white arrows) from an axonal bouton. Post-embedding immunoEM preparation reveals immunogold particles (black arrowheads) labeling NMDAR-GluN2B in the postsynaptic density. Adapted from .

Article Snippet: We performed a preadsorption control using the Alomone NMDAR-GluN2B blocking peptide (cat #BLP-GC003, ) and observed negligible labeling, suggesting that the regions outside the paratope of the antibody have minimal interactions in our tissue.

Techniques: Labeling

Journal: Frontiers in Neuroanatomy

Article Title: Contrasting patterns of extrasynaptic NMDAR-GluN2B expression in macaque subgenual cingulate and dorsolateral prefrontal cortices

doi: 10.3389/fnana.2025.1553056

Figure Lengend Snippet: Higher proportion of extrasynaptic GluN2B immunogold particles in layer III SGC spines than dlPFC spines. (A) Electron micrograph depicting spines in SGC layer III. (A1) An SGC spine (sp, pseudocolored yellow) with NMDAR-GluN2B immunogold particles (green arrowheads) in the postsynaptic density of a synapse (black arrows) formed by an axonal bouton (ax, pseudocolored blue). The spine contains a spine apparatus, an extension of the smooth endoplasmic reticulum (SER, pseudocolored pink) in the spine neck. (A2,A3) SGC spines with NMDAR-GluN2B appearing adhered to extrasynaptic membranes (orange arrowheads), near the SER. In A3, the extrasynaptic NMDAR-GluN2B is apposed to a structure consistent with glial morphology (gl, pseudocolored green), and the bouton contains a presynaptic cytoplasmic NMDAR-GluN2B (blue arrowhead) among the vesicles. (B) Electron micrographs depicting spines in dlPFC layer III. (B1,B2) dlPFC spines containing synaptic NMDAR-GluN2B (green arrowheads), and cytosolic NMDAR-GluN2B (gray arrowheads), which are likely being trafficked. (B3) A dlPFC spine with an extrasynaptic NMDAR-GluN2B in the spine neck, apposed to a structure consistent with glial morphology, and near a spine apparatus. (C) Nested pie charts depicting the location of NMDAR-GluN2B immunogold particles in spines of SGC (left), and dlPFC (right) in Monkey 1 (M1, inside) and Monkey 2 (M2, outside). Percent of NMDAR-GluN2B immunogold particles found in the cytoplasm (gray), post-synaptic density (green), perisynaptic membrane (yellow-green), and extrasynaptic membrane (orange) of NMDAR-GluN2B+ spines. (D) Plot depicting the location of membrane-bound NMDAR-GluN2B immunogold particles, in relation to the synapse, averaged across M1 and M2. Error bars depict standard deviation. One-way ANOVA, F (5,6 = 67.71, p < 0.001, with post-hoc Tukey’s test). * p < 0.05; ** p < 0.01, *** p < 0.001; **** p < 0.0001; scale bars, 200 nm. ax, axon; gl, glial process; mit, mitochondria; mvb, multivesicular body; SER, smooth endoplasmic reticulum spine apparatus; sp, spine.

Article Snippet: We performed a preadsorption control using the Alomone NMDAR-GluN2B blocking peptide (cat #BLP-GC003, ) and observed negligible labeling, suggesting that the regions outside the paratope of the antibody have minimal interactions in our tissue.

Techniques: Membrane, Standard Deviation

Journal: Frontiers in Neuroanatomy

Article Title: Contrasting patterns of extrasynaptic NMDAR-GluN2B expression in macaque subgenual cingulate and dorsolateral prefrontal cortices

doi: 10.3389/fnana.2025.1553056

Figure Lengend Snippet: Higher extrasynaptic expression in dendrites of putative excitatory neurons of SGC than dlPFC. (A) Electron micrographs from layer III SGC. (A1) A putative excitatory dendrite, labeled with MAP2+ non-nickel immunoperoxidase diaminobenzidine (smudge-like precipitate, double-headed arrows), expressing an extrasynaptic NMDAR-GluN2B (orange arrowhead); (A2,A3) NMDAR-GluN2B at extrasynaptic (orange arrowheads), cytoplasmic (gray arrowheads), or near-synaptic locations (gray-white striped arrowhead) in putative excitatory dendrites. (B) Electron micrographs from layer III dlPFC. (B1) A putative excitatory dendrite, with a spine in plane, expressing cytoplasmic and extrasynaptic NMDAR-GluN2B. (B2) A putative excitatory dendrite, labeled with MAP2, expressing extrasynaptic and cytoplasmic NMDAR-GluN2B. (C) Nested pie charts depicting the percent of NMDAR-GluN2B immunogold particles found at cytoplasmic, extrasynaptic, perisynaptic, and synaptic locations in MAP2+ dendritic shafts in SGC (left) and dlPFC (right) of Monkey 1 (inside) and Monkey 2 (outside). Synapses on the shaft of MAP2+ dendrites were rare, and synaptic NMDAR-GluN2B on MAP2+ shaft synapses was extremely rare (<0.2% of all immunogold particles in all areas analyzed). (D) Mean percent of extrasynaptic NMDAR-GluN2B immunogold particles across all MAP2+ dendrites in SGC and dlPFC. One-way ANOVA with post-hoc Tukey’s test, F (3,4) = 1,691, p < 0.0001. * p < 0.05; pink pseudocolor, SER spine apparatus; black arrows, synapse; scale bars, 200 nm; ax, axon; dend, dendrite; MAP2, microtubule-associated protein-2; mit, mitochondria; sp, spine.

Article Snippet: We performed a preadsorption control using the Alomone NMDAR-GluN2B blocking peptide (cat #BLP-GC003, ) and observed negligible labeling, suggesting that the regions outside the paratope of the antibody have minimal interactions in our tissue.

Techniques: Expressing, Labeling

Journal: Frontiers in Neuroanatomy

Article Title: Contrasting patterns of extrasynaptic NMDAR-GluN2B expression in macaque subgenual cingulate and dorsolateral prefrontal cortices

doi: 10.3389/fnana.2025.1553056

Figure Lengend Snippet: Extrasynaptic NMDAR-GluN2B expressed at a pyramidal-like soma in layer III SGC. (A) A panorama of stitched electron micrographs depicting a pyramidal-like soma and dendrites (pseudocolored yellow), including the nucleus (pseudocolored plum) and its axon initial segment (pseudocolored blue). NMDAR-GluN2B is prevalently expressed in extrasynaptic membranes (orange arrowheads) at the soma and proximal processes. Black boxes depict inset locations for subsequent panels. (B–G) Insets from (A) depicting extrasynaptic NMDAR-GluN2B at higher magnification. (H) Inset from (A) selected to emphasize the antibody labeling specificity of the labeled neuron compared to the surrounding neuropil as well as to emphasize nuclear labeling. Few NMDAR-GluN2B immunoparticles are evident in the surrounding neuropil, while the pyramidal-like soma densely expresses cytosolic NMDAR-GluN2B immunoparticles (gray arrowheads). NMDAR-GluN2B immunoparticles are also present in the nucleus (pseudocolored plum, white arrowhead). (I) Inset from (A) depicting several more extrasynaptic NMDAR-GluN2B on a basal dendrite as well as a symmetric synapse (double arrowheads) formed on the soma (axon pseudocolored red). Scale bars in (B–I) , 200 nm.

Article Snippet: We performed a preadsorption control using the Alomone NMDAR-GluN2B blocking peptide (cat #BLP-GC003, ) and observed negligible labeling, suggesting that the regions outside the paratope of the antibody have minimal interactions in our tissue.

Techniques: Antibody Labeling, Labeling

Journal: Frontiers in Neuroanatomy

Article Title: Contrasting patterns of extrasynaptic NMDAR-GluN2B expression in macaque subgenual cingulate and dorsolateral prefrontal cortices

doi: 10.3389/fnana.2025.1553056

Figure Lengend Snippet: NMDAR-GluN2B is expressed in inhibitory dendrites in layer III SGC and dlPFC. Electron micrographs depicting high-likelihood inhibitory dendrites in SGC (A) and dlPFC (B) . Dendrites were deemed high-likelihood inhibitory dendrites by the lack of spines in the plane and the presence of two or more asymmetric synapses formed on the dendritic shaft as inhibitory dendrites in the cortex are sparsely spiny or aspiny . (A1–A3) Examples of NMDAR-GluN2B immunogold labeling in high-likelihood inhibitory dendrites found in layer III SGC. (B1,B2) Examples of NMDAR-GluN2B immunogold labeling in high-likelihood inhibitory dendrites found in layer III dlPFC. (C) Nested pie charts depicting the location of NMDAR-GluN2B immunogold particles in high-likelihood dendrites of SGC (left) and dlPFC (right) for Monkey 1 (inside) and Monkey 2 (outside). No statistically significant differences were detected between dlPFC and SGC. Scale bars, 200 nm. ax, axon; dend, dendrite; gl, glial process; mit, mitochondria.

Article Snippet: We performed a preadsorption control using the Alomone NMDAR-GluN2B blocking peptide (cat #BLP-GC003, ) and observed negligible labeling, suggesting that the regions outside the paratope of the antibody have minimal interactions in our tissue.

Techniques: Labeling

Journal: Frontiers in Neuroanatomy

Article Title: Contrasting patterns of extrasynaptic NMDAR-GluN2B expression in macaque subgenual cingulate and dorsolateral prefrontal cortices

doi: 10.3389/fnana.2025.1553056

Figure Lengend Snippet: NMDAR-GluN2B are expressed at equivalent levels across CBP+ inhibitory neurons in layer III SGC and dlPFC. (A,B) Maximum projection images from confocal z-stacks obtained in layer III SGC (A) and dlPFC (B) depicting multiple immunofluorescence labeling for PV (red), CB (yellow), CR (magenta), and NMDAR-GluN2B (cyan). Color-coded arrows depict inhibitory neurons, and double-headed arrows indicate CB+ pyramidal neurons, which are faintly labeled and pyramidal in shape. (C,D) We sampled imaging fields in layer III of both SGC and the dlPFC for quantification of NMDAR-GluN2B expression across neurons labeled for the CBPs. Stacked bar charts depicting the mean proportion of PV (red), CB (yellow), and CR (magenta) that fell into negative, weak, moderate, or strong NMDAR-GluN2B expression bins as determined by the mean intensity (MI) in the NMDAR-GluN2B channel. “GluN2B-negative” is defined by an MI at or below the average amount in immunonegative sampled “background” neuropil regions. “GluN2B-strong” is defined by MI at or above the average found in nearby morphologically identified pyramidal-like neurons expressing NMDAR-GluN2B. See for more detailed information about the inhibitory neuron analysis that produced these plots. CB, calbindin; CR, calretinin; PV, parvalbumin.

Article Snippet: We performed a preadsorption control using the Alomone NMDAR-GluN2B blocking peptide (cat #BLP-GC003, ) and observed negligible labeling, suggesting that the regions outside the paratope of the antibody have minimal interactions in our tissue.

Techniques: Immunofluorescence, Labeling, Imaging, Expressing, Produced

Journal: Frontiers in Neuroanatomy

Article Title: Contrasting patterns of extrasynaptic NMDAR-GluN2B expression in macaque subgenual cingulate and dorsolateral prefrontal cortices

doi: 10.3389/fnana.2025.1553056

Figure Lengend Snippet: Schematic illustrating how extrasynaptic NMDAR-GluN2B may contribute to SGC hyperactivity and/or calcium-mediated neurodegeneration-related events. (A) Schematic depicting a spine (yellow), receiving a synapse from a glutamatergic bouton (blue), with an astrocytic leaflet (green) near the synapse. A synaptic NMDAR-GluN2B is present in the synapse (green), and an extrasynaptic NMDAR-GluN2B (orange) is present near the astrocytic process. (B) A magnified view showing that the bouton releases glutamate (gray circles) toward the postsynaptic density (thick black band), where the green synaptic NMDAR-GluN2B is engaged and calcium ions (pink), as well as sodium (Na+) ions, flow into the spine. AMPA receptors in the post-synaptic density also allow an influx of sodium ions (Na+), although these are emphasized less as they are not the focus of the present study. Incoming calcium ions can trigger feedforward calcium release ( ; ), via (1) direct calcium-mediated calcium release from the smooth endoplasmic reticulum (SER) by activation of primarily Ryanodine receptors (RyR), and (2) by cAMP magnification of calcium release, whereby calcium activates AC to produce cAMP, which activates PKA signaling. PKA, in turn, phosphorylates the SER calcium channels RyR and IP3R to further increase calcium release. Glutamate escaping out of the synaptic cleft is sequestered into the astrocyte via the EAAT, where it can be converted to glutamine. In the dlPFC, calcium influx can also lead to a reduction in delay-related firing via SK3 channels (not shown). (C) If the EAATs are perturbed or downregulated, then glutamate can more readily engage extrasynaptic NMDA-GluN2B. Evidence suggests that there may be glial pathology in the SGC during states of depression [ reviewed in , , , and ]. Given the prevalence of extrasynaptic NMDA-GluN2B, we have found in the present study, we hypothesize that these may contribute to SGC hyperactivity observed in depression, perhaps by engaging feedforward calcium mechanisms and increasing depolarization (yellow arrow). (D) Rapid-acting antidepressants that antagonize NMDA receptors may work in part by blocking extrasynaptic NMDAR-GluN2B in the SGC . (E) Calcium is normally tightly regulated by cytosolic buffering mechanisms (e.g., calbindin and mitochondria) and by phosphodiesterases (which catabolize cAMP). Loss of this regulation with aging and/or inflammation ( ; ; ) can dysregulate feedforward calcium signaling. Very high levels of cytosolic calcium can activate calpain-2, which cleaves and disinhibits GSK3β and cdk5, kinases that hyperphosphorylate tau, producing toxic species like pT217Tau . Further post-translational modifications lead to tau fibrillation and the formation of neurofibrillary tangles. AC, adenylyl cyclase; cAMP, cyclic adenosine monophosphate; cdk5, cyclin-dependent kinase 5; EAAT, excitatory amino acid transporter; GSK3β, glycogen synthase kinase 3 beta; IP3R, inositol triphosphate receptor; PKA, protein kinase A; SER, smooth endoplasmic reticulum spine apparatus; RyR, ryanodine receptor.

Article Snippet: We performed a preadsorption control using the Alomone NMDAR-GluN2B blocking peptide (cat #BLP-GC003, ) and observed negligible labeling, suggesting that the regions outside the paratope of the antibody have minimal interactions in our tissue.

Techniques: Activation Assay, Blocking Assay