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: Antioxidants

Article Title: The Application of the Neuroprotective and Potential Antioxidant Effect of Ergotamine Mediated by Targeting N-Methyl-D-Aspartate Receptors

doi: 10.3390/antiox11081471

Figure Lengend Snippet: ( A ) The chemical structure of ergotamine. ( B ) The H 2 O-injected oocytes did not cause any change with the treatment of 100 µM glutamate ( n = 6–8 oocytes from four different frogs). ( C – F ) Glutamate induced inward currents with or without ergotamine (30 µM and 10 µM). For each subunit of NMDARs, the responses after treating with either glutamate (100 µM) alone or together with ergotamine (30 and 10 µM). Voltage clamp recording was conducted at a holding potential of −80 mV. The coapplication of ergotamine with glutamate resulted in modulation of the recombinant receptors ( C ) NR1a/NR2A, ( D ) NR1a/NR2B, ( E ) NR1a/NR2C, and ( F ) NR1a/NR2D, which in turn reduced glutamate-evoked inward current in a reversible manner ( n = 6–8 oocytes from four different frogs).

Article Snippet: The mouse NMDAR subunit cDNAs included the NR1 (GenBank accession number: MR225704), NR2A (MR227135), NR2B (MR227077), NR2C (MR222676), and NR2D (MR220972) subunits, which were purchased from OriGene (Rockville, MD, USA).

Techniques: Injection, Recombinant

Journal: Antioxidants

Article Title: The Application of the Neuroprotective and Potential Antioxidant Effect of Ergotamine Mediated by Targeting N-Methyl-D-Aspartate Receptors

doi: 10.3390/antiox11081471

Figure Lengend Snippet: The value of I max , IC 50 , and n H (Hill coefficient) of ergotamine for the glutamate-evoked current in each recombinant receptor. Values represent means ± S.E.M. ( n = 6–8/group). IC 50 , Hill’s coefficient; I max value as determined as described in Materials and methods.

Article Snippet: The mouse NMDAR subunit cDNAs included the NR1 (GenBank accession number: MR225704), NR2A (MR227135), NR2B (MR227077), NR2C (MR222676), and NR2D (MR220972) subunits, which were purchased from OriGene (Rockville, MD, USA).

Techniques: Recombinant

Journal: Molecular Pain

Article Title: NMDA Receptor-Dependent Synaptic Depression in Potentiated Synapses of the Anterior Cingulate Cortex of adult Mice

doi: 10.1177/17448069211018045

Figure Lengend Snippet: GluN2C/D-containing NMDA receptors were not involved in synaptic depression after synaptic potentiation. (a) The summarized fEPSP slope plot about the effect of PPDA (GluN2C/D-selective NMDA receptors antagonist, 10 µM) on synaptic depression after potentiation (n = 9 slices/8 mice). (b) Statistical results showed that significant difference was observed between TBS and LFS by comparisons of the last 10 min fEPSP slopes in presence of PPDA (TBS: 130.48 ± 2.80% of the baseline; LFS: 102.98 ± 6.17%). ** p <0.01, paired t -test, compared with TBS. (c) The averaged fEPSP slope plot about the effect of UBP145 (GluN2D-selective NMDA receptors antagonist, 3 µM) on synaptic depression after potentiation (n = 14 slices/11 mice). (d) Statistical results showed that the averaged fEPSP slope after TBS was higher than that of LFS (TBS: 123.94 ± 1.77%; LFS: 114.43 ± 3.56% of the baseline; * p <0.05, paired t -test, compared with TBS). Error bars indicated SEM.

Article Snippet: Selective competitive NMDA receptor antagonist AP-5 (Cat. No. HB0225) and GluN2D-selective NMDA receptor antagonist UBP145 (Cat. No. HB4717) were purchased from HelloBio (Princeton, NJ, USA).

Techniques:

Journal: Neuropharmacology

Article Title: Multiple roles of GluN2D-containing NMDA receptors in short-term potentiation and long-term potentiation in mouse hippocampal slices

doi: 10.1016/j.neuropharm.2021.108833

Figure Lengend Snippet: Comparison of baseline synaptic transmission and PPF of WTs and GluN2D KOs. (A) FV amplitude plotted against stimulus intensity. Slopes of WT line (−0.048 mV/stim intensity, n = 14) and KO line (−0.051 mV/stim intensity, n = 13) were not significantly different (F(1, 239) = 0.5774, p = 0.448, F test). (B) fEPSP to FV relationship for WT (slope of line = 1.8 ms −1 , n = 15) and KO (slope of line = 1.9 ms −1 , n = 11) were not significantly different (F(1, 15) = 0.9132, p = 0.3544, F test). (C) PPF (50 ms inter-pulse interval) was not significantly different between WTs (2.1 ± 0.07, n = 15) and KOs (2.1 ± 0.06, n = 15, t(28) = 0.2460, p = 0.8075, t -test). (D) Representative fEPSP traces from points indicated in A. (E) Representative fEPSP traces showing PPF in WTs (black) and GluN2D KOs (grey).

Article Snippet: The global GluN2D KO mice were obtained from Professor Masayoshi Mishina (University of Kyoto) and maintained at a breeding facility in the UK (Charles River).

Techniques: Comparison, Transmission Assay

Journal: Neuropharmacology

Article Title: Multiple roles of GluN2D-containing NMDA receptors in short-term potentiation and long-term potentiation in mouse hippocampal slices

doi: 10.1016/j.neuropharm.2021.108833

Figure Lengend Snippet: Altered synaptic plasticity in GluN2D KOs. (A) Pooled data showing the time course of potentiation of fEPSPs (mean ± SEM) for WTs (filled circles, n = 21) and KOs (open circles, n = 17). Decay of STP was fitted using a bi-exponential decay function (black curve fits WT and grey curve fits KO). The rate of decay of STP1 and STP2 were not significantly different between WTs (τ 1 = 3.0 min, τ2 = 24.0 min) and KOs (τ 1 = 4.7 min, τ2 = 25.8 min) (τ 1 F(1, 3333) = 1.231, p = 0.267, τ 2 F(1, 3333) = 0.0801, p = 0.777, F test). In this and subsequent time course plots, the arrowhead indicates the time of high frequency stimulation and the associated “B” refers to the number of bursts delivered. Representative fEPSPs from WTs and KOs at the time-points indicated in A are shown on the right. (B) The level of STP1 and STP2 were calculated by integrating the fast and slow components of the bi-exponential decay function, respectively, and are presented normalised with respect to the corresponding control. STP1 was significantly lower in WTs (100.0 ± 10.1%) compared to KOs (212.4 ± 19.4%; ****p < 0.0001, t(36) = 5.431, t -test). STP2 was not significantly different between WTs (100.0 ± 13.0%) and KOs (138.7 ± 15.7%; p = 0.0633, t(36) = 1.916, t -test). LTP in WTs (38.5 ± 4.3%) was significantly less than in KOs (52.5 ± 5.5%; t(36) = 2.055, *p = 0.0472, t -test) (C) Time course of potentiation in WTs (filled circles, n = 4) and KOs (open circles, n = 5) in the presence of GABA A and GABA B receptor antagonists. Decay time constant of STP1 and STP2 were not different between WTs (τ 1 = 1.7 min, τ 2 = 19.2 min) and KOs (τ 1 = 2.1 min, τ 2 = 25.1 min; τ 1 F(1, 796) = 1.081, p = 0.299; τ 2 F(1, 796) = 1.172, p = 0.279, F test). In this and subsequent figures, horizontal bars on the time course plots indicates the duration of compound application. (D) STP1 level was significantly lower in WT (100 ± 8.8%) compared to KOs (191.6 ± 32.2%; *p = 0.0437, t(7) = 2.456, t -test). STP2 was significantly greater in WTs (100 ± 13.4%) compared to KOs (45.5 ± 14.5%; *p = 0.0309, t(7) = 2.694, t -test). LTP was similar in WTs (79.4 ± 8.1%) and KOs (67.4 ± 10.7%; t(7) = 0.8548, p = 0.421; t -test).

Article Snippet: The global GluN2D KO mice were obtained from Professor Masayoshi Mishina (University of Kyoto) and maintained at a breeding facility in the UK (Charles River).

Techniques: Control

Journal: Neuropharmacology

Article Title: Multiple roles of GluN2D-containing NMDA receptors in short-term potentiation and long-term potentiation in mouse hippocampal slices

doi: 10.1016/j.neuropharm.2021.108833

Figure Lengend Snippet: Enhanced STP and LTP in GluN2D KOs is NMDAR-dependent. (A) Time course of STP and LTP by 10-bursts in WTs. Data from control experiments (Ctrl, black circles, n = 8), 100 μM D-AP5 (red circles, n = 5) and 10 μM L-689,560 (blue circles, n = 3) are shown. (B) Summary of STP1, STP2 and LTP in WTs (from experiments shown in A) from control (STP1 = 100.0 ± 12.3%, STP2 = 100.0 ± 17.5%, LTP = 40.2 ± 8.0%), D-AP5 (STP1 = 3.1 ± 3.1%, STP2 = 50.3 ± 12.0%, LTP = 5.4 ± 2.4%) and L-689,560 (STP1 = 7.4 ± 7.4%, STP2 = 20.7 ± 10.5%, LTP = 2.0 ± 1.1%). STP1 was inhibited by D-AP5 (t(13) = 6.475, ****p < 0.0001, ANOVA with Bonferroni test for multiple comparisons (BT)) and L-689,560 (t(13) = 5.208, ***p = 0.0003, ANOVA with BT). STP2 was not significantly different in the D-AP5 (t(13) = 2.193, p = 0.0941, ANOVA) group but was significantly reduced in L-689,560 (t(13) = 2.945, *p = 0.0227, ANOVA with BT), compared to control. LTP was inhibited by D-AP5 (t(13) = 3.607, **p = 0.0064, ANOVA with BT) and L-689,560 (t(13) = 3.332, *p = 0.0108, ANOVA with BT). (C) Similar to A, but data are from KOs. Interleaved controls (grey circles, n = 8), D-AP5 (red circles, n = 6), and L-689,560 (blue circles, n = 4). (D) Summary of STP and LTP in KOs (from experiments shown in C) from control (STP1 = 100.0 ± 18.2%, STP2 = 100.0 ± 21.1%, LTP = 47.9 ± 8.8%), D-AP5 (STP1 = 17.9 ± 9.8%, STP2 = 34.1 ± 11.0%, LTP = 7.1 ± 3.0%) and L-689,560 (STP1 = 12.2 ± 3.3%, STP2 = 7.6 ± 2.7%, LTP = 5.2 ± 1.4%) experiments. STP1 was inhibited by D-AP5 (t(15) = 4.003, **p = 0.0023, ANOVA with BT) and L-689,560 (t(15) = 3.776, **p = 0.0037, ANOVA with BT). STP2 was inhibited by D-AP5 (t(15) = 2.791, *p = 0.0274, ANOVA with BT) and L-689,560 (t(10) = 3.451, **p = 0.007, ANOVA with BT), compared to control. LTP was inhibited by D-AP5 (t(15) = 4.280, **p = 0.0013, ANOVA with BT) and L-689,560 (t(15) = 3.956, **p = 0.0025, ANOVA with BT) compared to control.

Article Snippet: The global GluN2D KO mice were obtained from Professor Masayoshi Mishina (University of Kyoto) and maintained at a breeding facility in the UK (Charles River).

Techniques: Control

Journal: Neuropharmacology

Article Title: Multiple roles of GluN2D-containing NMDA receptors in short-term potentiation and long-term potentiation in mouse hippocampal slices

doi: 10.1016/j.neuropharm.2021.108833

Figure Lengend Snippet: UBP145 partially inhibits LTP in WTs but has no effect in GluN2D KOs. (A) Time course of potentiation induced by 10-bursts in the presence of GluN2D antagonist 10 μM UBP145 (green circles, n = 7) compared to controls (black circles, n = 7) in WTs. (B) Summary of STP1, STP2 and LTP in WTs (from experiments shown in A) in control (STP1 = 100.0 ± 12.0%, STP2 = 100.0 ± 18.4%, LTP = 47.5 ± 8.3%) and UBP145 experiments (STP1 = 91.6 ± 18.4%, STP2 = 61.6 ± 20.3%, LTP = 23.1 ± 6.5%) in WTs. STP1 was not significantly different between the control and UBP145 groups (t(12) = 0.3813, p = 0.7096, t -test). STP2 was not significantly different between control and UBP145 (t(12) = 1.401, p = 0.1864, t -test). LTP was significantly reduced in UBP145-treated group compared to control (t(12) = 2.314, *p = 0.0392, t -test). (C) GluN2D KOs treated with UBP145 (green circles, n = 8) and controls (grey circles, n = 8). (D) Summary of potentiation in control (STP1 = 100.0 ± 15.8%, STP2 = 100.0 ± 12.4%, LTP = 46.8 ± 9.1%) and UBP145 experiments (STP1 = 71.8 ± 11.1%, STP2 = 83.8 ± 25.6%, LTP = 44.4 ± 7.5%) in KOs. STP1, STP2 and LTP were not significantly different between the control and UBP145 groups in KOs (STP1: t(14) = 1.458, p = 0.1668, t -test; STP2: t(14) = 0.5712, p = 0.5769, t -test; LTP: t(14) = 0.2077, p = 0.8384, t -test).

Article Snippet: The global GluN2D KO mice were obtained from Professor Masayoshi Mishina (University of Kyoto) and maintained at a breeding facility in the UK (Charles River).

Techniques: Control

Journal: Neuropharmacology

Article Title: Multiple roles of GluN2D-containing NMDA receptors in short-term potentiation and long-term potentiation in mouse hippocampal slices

doi: 10.1016/j.neuropharm.2021.108833

Figure Lengend Snippet: The GluN2D subunit contributes to STP2 and LTP. (A) Time course of potentiation in WTs induced by 30B in controls (purple symbols, n = 4), or in the presence of 10 μM UBP145 (green symbols, n = 4). (B) Summary of STP1, STP2 and LTP in WTs (from experiments shown in A) in control (STP1 = 100.0 ± 31.0%, STP2 = 100.0 ± 17.2%, LTP = 84.3 ± 10.7%) and UBP145 (STP1 = 92.9 ± 27.8%, STP2 = 30.6 ± 18.0%, LTP = 48.5 ± 10.0%) experiments in WTs. STP1 was not significantly different in UBP145 (t(6) = 0.1719, p = 0.8692, t -test) compared to control. STP2 was significantly lower in UBP145 (t(6) = 2.793, *p = 0.0315, t -test). LTP was partially inhibited by UBP145 (t(6) = 3.405, *p = 0.0144, t -test). (C) Equivalent data from KOs. Potentiation of control KOs (purple symbols, n = 6), UBP145 (green symbols, n = 6). (D) Summary of potentiation in control (STP1 = 100.0 ± 31.0%, STP2 = 100.0 ± 9.0%, LTP = 56.8 ± 6.0%), UBP145 (STP1 = 134.4 ± 35.4%, STP2 = 89.4 ± 16.0%, LTP = 51.3 ± 7.7%). STP1, STP2 and LTP were not significantly different upon treatment with UBP145 (STP1: t(10) = 0.732, p = 0.4811, STP2: t(10) = 0.575, p = 0.5781, LTP t(10) = 0.561, p = 0.5869, t -test).

Article Snippet: The global GluN2D KO mice were obtained from Professor Masayoshi Mishina (University of Kyoto) and maintained at a breeding facility in the UK (Charles River).

Techniques: Control

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

Journal: Frontiers in Synaptic Neuroscience

Article Title: GluN2D NMDA Receptors Gate Fear Extinction Learning and Interneuron Plasticity

doi: 10.3389/fnsyn.2021.681068

Figure Lengend Snippet: Deletion of GluN2D subunits abolishes I-LTD stim in conditioned mice. (A) Representative traces of mIPSCs in stellate cells from naïve ( top ) and paired ( bottom ) GluN2D KO mice. (B) Individual frequencies and amplitudes of mIPSCs in naïve (open red circle, n = 10) and paired mutant animals (filled red circles, n = 11). (C) Fear conditioning protocol for electrophysiology experiments. (D) Schematic of the experimental procedure. (E) Left , Example traces of mIPSCs recorded in MLIs before ( top ) and after ( bottom ) parallel fiber stimulation (15 trains of four pulses at 100 Hz) in GluN2D KO mice after fear conditioning. Right , corresponding time course of mIPSC frequency (top) and amplitude (bottom). (F) Average time course of mIPSC frequency normalized to before parallel fiber stimulation in GluN2D KO mice (red circles, values are mean ± SEM) after fear learning. Wildtype naïve and paired group average values (blue lines) and SEM (light blue area) are data from represented for reference. (G) Summary of the individual frequencies ( left ) and amplitudes ( right ) before ( Pre ) and 15–30 min after parallel fiber stimulation ( Post ). ** P < 0.01. Statistical analysis values can be found in the .

Article Snippet: These animals were either wildtype (Jackson laboratory Bar Harbor, ME, United States) or GluN2D KO mice ( ; ).

Techniques: Mutagenesis

Journal: Frontiers in Synaptic Neuroscience

Article Title: GluN2D NMDA Receptors Gate Fear Extinction Learning and Interneuron Plasticity

doi: 10.3389/fnsyn.2021.681068

Figure Lengend Snippet: Deletion of GluN2D does not alter fear conditioning learning or memory retention but abolishes extinction learning. (A) Protocol used for fear conditioning. Mice were habituated for 2 min in the conditioning chamber (context A) and exposed to eight pairings of a 10 s tone that co-terminated with a 1 s foot-shock. Mice were then left in the conditioning chamber for 2 min and returned to their home cage. Next day mice were exposed to a single 10 s tone in context B. (B) Percentage of freezing in wildtype (blue symbols, n = 10) and GluN2D KO mice (red symbols, n = 8) during the habituation period (H), the conditioning training (tones 1–8) and retention test tone. (C) Freezing time in individual animals during fear memory retention test. Mean values are represented as doted lines. (D) A two pairing conditioning paradigm was used. Next day retention and extinction learning (Ext tr) were tested in context B by exposing the animals to eight 10 s tones after 2 min of habituation. (E) GluN2D KO mice (red symbols, n = 11), but not wildtype (blue symbols, n = 11), showed impaired extinction learning assessed on the last tone. (F) Freezing time in individual animals at the end of extinction training. Mean values are represented as doted lines. *** P < 0.001. Values in the time courses are mean ± SEM. Statistical analysis values can be found in the .

Article Snippet: These animals were either wildtype (Jackson laboratory Bar Harbor, ME, United States) or GluN2D KO mice ( ; ).

Techniques:

Journal: Frontiers in Synaptic Neuroscience

Article Title: GluN2D NMDA Receptors Gate Fear Extinction Learning and Interneuron Plasticity

doi: 10.3389/fnsyn.2021.681068

Figure Lengend Snippet: D-cycloserine fails to rescue extinction learning in GluN2D KO mice. (A) Wildtype and GluN2D KO mice were injected with D-cycloserine (DCS, 10 mg/kg; i.p.) or saline (Sal), 30 min before fear extinction learning. (B) Freezing response in wildtype mice injected with either saline (blue symbols, n = 9) or D-cycloserine (yellow symbols, n = 8) showed that D-cycloserine administration significantly accelerated extinction learning on tones 4–6. (C) Individual values for freezing response during extinction training. (D) Freezing response in GluN2D KO mice injected with either saline (red symbols, n = 12) or D-cycloserine (yellow symbols, n = 13) showed that D-cycloserine administration failed to accelerate extinction learning. (E) Individual freezing values at the end of the extinction training. * P < 0.05. Values are mean ± SEM. Statistical analysis values can be found in the .

Article Snippet: These animals were either wildtype (Jackson laboratory Bar Harbor, ME, United States) or GluN2D KO mice ( ; ).

Techniques: Injection, Saline

Journal: Frontiers in Synaptic Neuroscience

Article Title: GluN2D NMDA Receptors Gate Fear Extinction Learning and Interneuron Plasticity

doi: 10.3389/fnsyn.2021.681068

Figure Lengend Snippet: Retrieval-extinction paradigm fails to rescue extinction learning in GluN2D KO mice. (A) Protocol used for retrieval-extinction paradigm. Following fear conditioning, mice were exposed to a retrieval tone in context B on day 2. Thirty min later mice underwent two extinction sessions of 20 tones 30 min apart. On day 3, mice were presented with four tones in context B to test for the retention of extinction memory. (B) Percentage of freezing in wildtype (blue symbols, n = 10) and GluN2D KO mice (red symbols, n = 7). Wild type mice exhibited a clear extinction learning. Extinction learning in GluN2D KO mice was attenuated compared with wild type animals. (C) Individual freezing values at the end of extinction training. Values in the time course are mean ± SEM. * P < 0.05. Statistical analysis values can be found in the .

Article Snippet: These animals were either wildtype (Jackson laboratory Bar Harbor, ME, United States) or GluN2D KO mice ( ; ).

Techniques:

Journal: Frontiers in Synaptic Neuroscience

Article Title: GluN2D NMDA Receptors Gate Fear Extinction Learning and Interneuron Plasticity

doi: 10.3389/fnsyn.2021.681068

Figure Lengend Snippet: Summary schematic. Top , the behavioral response of wildtype and GluN2D KO mice during fear conditioning and extinction training. Bottom , correlated cellular events at the stellate-to-stellate cell synapse in the cerebellar cortex. The bottom part of the schematic was created with biorender.com .

Article Snippet: These animals were either wildtype (Jackson laboratory Bar Harbor, ME, United States) or GluN2D KO mice ( ; ).

Techniques: