Journal: Channels

Article Title: Differential expression of GluN2 NMDA receptor subunits in the dorsal horn of male and female rats

doi: 10.1080/19336950.2020.1871205

Figure Lengend Snippet: List of antibodies used for immunohistochemistry

Article Snippet: Rabbit anti-GluN2D , 1:1000 , Alomone Labs , ACG-020.

Techniques:

Journal: Channels

Article Title: Differential expression of GluN2 NMDA receptor subunits in the dorsal horn of male and female rats

doi: 10.1080/19336950.2020.1871205

Figure Lengend Snippet: Immunolabelling of GluN2A, GluN2B and GluN2D subunits in the SDH and DDH of male and female rats. The CGRP+ marginal region of the SDH as well as the remaining DDH was outlined as described in . For immunoreactive quantification in subsequent figures, SDH and DDH staining (per area) were normalized to background (BG) signal in a boxed region of the immuno-negative dorsal column. A, bottom) Representative confocal images (20x objective) illustrating the fluorescence staining patterns for GluN2A (left), GluN2B (middle) and GluN2D (right) in male juvenile rats. Scale bar: 200 μm. A, top) Higher magnification insets illustrating the distribution of immunoreactivity in the marginal SDH for all three GluN2 subtypes. GluN2A immunoreactivity is uniformly distributed across the neuropil of the SDH and DDH laminae, while GluN2B and GluN2D signal is also widespread but with differential expression in SDH versus DDH laminae, with staining of both neuropil and cell bodies. Scale bar: 50 µm. B, bottom) Representative confocal images (20x objective) illustrating the fluorescence staining patterns for GluN2A (left), GluN2B (middle) and GluN2D (right) in female juvenile rats. Scale bar: 200 μm. A, top) Higher magnification insets illustrating the distribution of immunoreactivity in the marginal SDH for all three GluN2 subtypes. GluN2A immunoreactivity is distributed mainly in the neuropil of the SDH and DDH laminae, while GluN2B and GluN2D signal demonstrates staining of both neuropil and cell bodies in the SDH and DDH. Scale bar: 50 µm

Article Snippet: Rabbit anti-GluN2D , 1:1000 , Alomone Labs , ACG-020.

Techniques: Staining, Fluorescence, Expressing

Journal: Channels

Article Title: Differential expression of GluN2 NMDA receptor subunits in the dorsal horn of male and female rats

doi: 10.1080/19336950.2020.1871205

Figure Lengend Snippet: Relative expression of GluN2A, GluN2B and GluN2D in the SDH versus the DDH of males and female rats. Optical density of a given GluN2 signal per area for the SDH as well as for the DDH was normalized to background optical density per area (see box in ). Average SDH and DDH measures were derived from multiple spinal sections for each animal (shown as individual data points), followed by a paired comparison between the normalized SDH and DDH for each GluN2 subtype. (a) In males, there is significantly higher expression in the SDH (dark blue bars) versus the DDH (light blue bars) for all the three GluN2 subunits, but with more pronounced differential expression for GluN2B (middle) and GluN2D (right). (b) In females, the GluN2B isoform (middle) is the only GluN2 subunit that exhibits significantly greater expression in the SDH (dark purple) versus the DDH (light purple). *p < 0.05

Article Snippet: Rabbit anti-GluN2D , 1:1000 , Alomone Labs , ACG-020.

Techniques: Expressing, Derivative Assay

Journal: Channels

Article Title: Differential expression of GluN2 NMDA receptor subunits in the dorsal horn of male and female rats

doi: 10.1080/19336950.2020.1871205

Figure Lengend Snippet: The GluN2B, but not GluN2A or GluN2D, isoform is preferentially expressed in the medial versus the lateral region of the SDH in male rats. (a) Representative confocal images (20x objective) illustrating the immunofluorescence for GluN2A (left), GluN2B (middle), and GluN2D (left). For each isoform, the OD per area was quantified in selected oval regions positioned within the medial, central and lateral regions of the CGRP+ SDH, as shown. There was a visible increase in intensity and distribution of fluorescent puncta in the medial SDH compared to the lateral SDH for GluN2B but not GluN2A or GluN2D. Scale bar: 200 µm. (b) Statistical analysis showing the mean medial vs lateral OD ratio for GluN2A, GluN2B and GluN2D. The medial/lateral OD ratio was significantly higher for GluN2B compared to both GluN2A and GluN2D. *p < 0.05

Article Snippet: Rabbit anti-GluN2D , 1:1000 , Alomone Labs , ACG-020.

Techniques: Immunofluorescence

Journal: Channels

Article Title: Differential expression of GluN2 NMDA receptor subunits in the dorsal horn of male and female rats

doi: 10.1080/19336950.2020.1871205

Figure Lengend Snippet: Comparison of the medial vs lateral OD ratio of GluN2 subunit isoforms between males and female rats. A, B) Representative confocal images showing GluN2B immunofluorescence in a male (a) and female rat (b), Scale bar: 200 μm, as well as higher magnification insets of the SDH for both male (top left) and female (top right), Scale bar: 50 μm. GluN2B signal is preferentially localized to the medial portion of the SDH in males (a) but not females (b). (c–e) Quantitative statistical analysis of the mean medial vs lateral OD ratio for GluN2A (c), GluN2B (d) and GluN2D (e) in males versus females. The medial/lateral ratio for GluN2B was significantly greater in males compared to females (d), while the medial/lateral ratio was not significantly different between males and females and near values of 1 for both GluN2A (c) and GluN2D (e). *p < 0.05

Article Snippet: Rabbit anti-GluN2D , 1:1000 , Alomone Labs , ACG-020.

Techniques: Immunofluorescence

Journal: British Journal of Pharmacology

Article Title: Ketamine and phencyclidine: the good, the bad and the unexpected

doi: 10.1111/bph.13222

Figure Lengend Snippet: Tables of Links

Article Snippet: [ PubMed ] Yamamoto H, Kamegaya E, Sawada W, Hasegawa R, Yamamoto T, Hagino Y, et al. Involvement of the N-methyl-D-aspartate receptor GluN2D subunit in phencyclidine-induced motor impairment, gene expression, and increased Fos immunoreactivity.

Techniques:

Journal: The Journal of Neuroscience

Article Title: GluN2D Subunit-Containing NMDA Receptors Control Tissue Plasminogen Activator-Mediated Spatial Memory

doi: 10.1523/JNEUROSCI.6202-11.2012

Figure Lengend Snippet: Active immunization selectively prevents tPA/GluN1 interaction and tPA-induced enhancement of NMDAR signaling without altering the basal Ca2+ conductivity. A, Schematic representation of NMDAR composed of GluN1/GluN2D subunits, including binding sites of αATD–GluN1, αCTD–GluN1, and αCTD–GluN2D antibodies, UBP145, and tPA. B, ATD–GluN1 immunized mice display antibodies specifically targeting the GluN1 subunit of NMDAR. Proteins extracts from naive mouse brain (n = 3) were subjected to immunoblots revealed with IgGs purified from either control mice (control IgGs) or ATD–GluN1 (120 kDa) immunized mice (αATD–GluN1). Parallel immunoblottings were performed and revealed with antibodies raised against either CTD–GluN1 (named αCTD–GluN1), known to reveal a band at ∼120 kDa, or CTD–GluN2D (named αCTD–GluN2D), known to reveal a band at ∼165 kDa. C, After immunization, mice display circulating antibodies against GluN1, capable of preventing the potentiating effect of tPA on GluN1/GluN2D subunit-containing NMDARs. NMDA induces Ca2+ influx in cortical neurons as measured by fura-2 video microscopy (N = 3, n = 150 cells). Coapplication of tPA (20 μg/ml; 45 min) potentiates the NMDA-evoked Ca2+ influx by 47% (N = 3, n = 108 cells). Neither UBP145 alone (0.2 μm; N = 3, n = 150 cells) nor αATD–GluN1 antibodies alone (0.01 mg/ml; N = 3, n = 108 cells) alter NMDA-induced Ca2+ influx. Both UBP145 (0.2 μm) and αATD–GluN1 (0.01 mg/ml) are capable of blocking this potentiating effect of tPA (N = 3, n = 150 cells and N = 3, n = 108 cells, respectively). Ctrl, Control; HBBSS, serum-free medium. Paired Student's t test (before vs after treatment), *p < 0.001. Vertical bars indicate SD.

Article Snippet: Homozygous male mutant mice lacking the GluN2D subunit of NMDAR were generated by Prof. Mishina (University of Tokyo, Tokyo, Japan) ( Ikeda et al., 1995 ) and provided by the RIKEN BioResource Center.

Techniques: Binding Assay, Western Blot, Purification, Control, Microscopy, Blocking Assay

Journal: The Journal of Neuroscience

Article Title: GluN2D Subunit-Containing NMDA Receptors Control Tissue Plasminogen Activator-Mediated Spatial Memory

doi: 10.1523/JNEUROSCI.6202-11.2012

Figure Lengend Snippet: Regulation of NMDAR subunits (GluN1, GluN2A, GluN2B, GluN2D) in hippocampus after active immunization against the ATD of the NMDAR GluN1 subunit. Relative mRNA quantity, estimated by RT-qPCR, was expressed in 2−(Ct gene of interest), in which Ct is the threshold cycle value. A, GluN1 subunit mRNA expression. B, GluN2A subunit mRNA expression. C, GluN2B subunit mRNA expression. D, GluN2D subunit mRNA expression. WT Crude ATD mice, n = 5; WT Crude Control mice, n = 5. Mann–Whitney U test, *p < 0.05. Vertical bars indicate SD.

Article Snippet: Homozygous male mutant mice lacking the GluN2D subunit of NMDAR were generated by Prof. Mishina (University of Tokyo, Tokyo, Japan) ( Ikeda et al., 1995 ) and provided by the RIKEN BioResource Center.

Techniques: Quantitative RT-PCR, Expressing, Control, MANN-WHITNEY

Journal: The Journal of Neuroscience

Article Title: GluN2D Subunit-Containing NMDA Receptors Control Tissue Plasminogen Activator-Mediated Spatial Memory

doi: 10.1523/JNEUROSCI.6202-11.2012

Figure Lengend Snippet: GluN1/GluN2D subunit-containing NMDARs drive tPA-influenced spatial memory. A, Previous studies have evidenced that tPA was not involved in locomotor activity (Pawlak et al., 2002). However, tPA is known to influence both emotional (Calabresi et al., 2000) and spatial memories (Benchenane et al., 2007). B, Our present experiments reveal that inhibition of the tPA/NMDAR interaction prevents neither locomotor activity nor emotional memory in mice. In addition, our results show that the tPA/NMDAR interaction is a critical mechanism underlying tPA-influenced spatial memory. C, In agreement with Ikeda et al. (1995), we observe a decrease in spontaneous locomotor activity in GluN2D-deficient mice. Furthermore, our present study reveals impairments of both emotional and spatial memories in this strain. In addition, we also show that the inhibition of tPA/NMDAR interaction does not impair the spatial memory in GluN2D KO mice. Together, these results demonstrate that tPA influences spatial memory through an increased affinity for NMDAR when associated with GluN2D subunit. Arrows point to the behavioral deficit.

Article Snippet: Homozygous male mutant mice lacking the GluN2D subunit of NMDAR were generated by Prof. Mishina (University of Tokyo, Tokyo, Japan) ( Ikeda et al., 1995 ) and provided by the RIKEN BioResource Center.

Techniques: Activity Assay, Inhibition