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: bioRxiv

Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

doi: 10.1101/2020.06.03.130559

Figure Lengend Snippet: (A-E) Postnatal development of callosal projection in S1. (A) EGFP plasmid injected into lateral ventricle of embryo at embryonic day15.5 (E15.5) and electrical pulse given to enable the plasmid to enter cortical progenitor cells of layer II/III in the ventricular zone. (B, B’) At postnatal day 5 (P5), the callosal axons from S1 had reached the white matter underneath contralateral S1. (C, C’) At P8, the callosal axons were diffusely distributed in contralateral S1. (D, D’) By P12, pruning of excess projections led to a refined innervation pattern with a narrow band limited to the S1/S2 border. (E, E’) After P12, the pattern was stable as observed at P30. (F) In P14 control mice ( Emx1 cre/+ ; NR1 fl/wt ), the callosal innervation pattern of S1 of the contralateral cortex is well-differentiated with a dense innervation at S1/S2 border. The pattern persists to P30 (J). (G) In NR1 KO mice ( Emx1 cre/+ ; NR1 fl/fl ), the innervation pattern was disrupted and projections were extremely diffuse which also persisted to P30 (K). (H) Quantification of fluorescent intensity across the medial to lateral extent of the S1. (I, L) Quantification of fluorescence density of S1 region of control vs. NR1 KO mice at P14 (I, P = 0.002) and P30 (L, P = 0.0003) Scale bar: 500μm for all images. S1: primary somatosensory cortex; S2: secondary somatosensory cortex.

Article Snippet: Antibodies for intraventricular injection : commercial anti-NMDAR antibody is against amino acid residues 385-399 in the N-terminus of NR1 and was made in Rabbit (AGC-001, Alomone labs).

Techniques: Plasmid Preparation, Injection, Control, Fluorescence

Journal: bioRxiv

Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

doi: 10.1101/2020.06.03.130559

Figure Lengend Snippet: Examples of 12-μm coronal brain sections from P8 Emx1 cre/+ ; NR1 wt/wt (A) and Emx1 cre/+ ; NR1 fl/fl (B) of the same litter. Immunostaining of vesicular glutamate transporter 2 (VGult2) showed thalamocortical barrels in Layer IV of S1 which are pointed out by arrows. The VGlut2 staining in Emx1 cre/+ ; NR1 wt/wt mice revealed a clear barrel pattern (Aa). However, the barrel pattern in Emx1 cre/+ ; NR1 fl/fl mice was disrupted and less distinct (Ba). The NR1 staining in Emx1 cre/+ ; NR1 wt/wt mice were dense and strong in cortex (Ab, Ac). However, the staining in Emx1 cre/+ ; NR1 fl/fl mice was less bright and apparently reduced in Layer V and VI (Bb, Bc). Scale bar: 100μm for Ac and Bc; 500μm for rest of images.

Article Snippet: Antibodies for intraventricular injection : commercial anti-NMDAR antibody is against amino acid residues 385-399 in the N-terminus of NR1 and was made in Rabbit (AGC-001, Alomone labs).

Techniques: Immunostaining, Staining

Journal: bioRxiv

Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

doi: 10.1101/2020.06.03.130559

Figure Lengend Snippet: (A, B) The callosal axons in S1 formed a bundle and grew into the ipsilateral CC at P0 in control and NR1 KO littermates ( Emx1 cre/+ ; NR1 fl/wt and Emx1 cre/+ ; NR1 fl/fl mice). The arrows show the extent of axon growth into the CC. By P3, the callosal axons crossed the midline (C, D) and by P5, the callosal axons have grown to underneath the contralateral S1 (E, F). Scale bar: 500μm for all images.

Article Snippet: Antibodies for intraventricular injection : commercial anti-NMDAR antibody is against amino acid residues 385-399 in the N-terminus of NR1 and was made in Rabbit (AGC-001, Alomone labs).

Techniques: Control

Journal: bioRxiv

Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

doi: 10.1101/2020.06.03.130559

Figure Lengend Snippet: (A, A’) At P6, most axons in control grew into deeper layer VI of S1 (see “*”); a few axons grew to layer V from medial to lateral S1 (see arrows). However, axons projecting to lateral S2 had grown to layer IV which was apparently faster than the axons in S1 (see arrows). (B, B’) In NR1 KO mice, most axons had grown to layer V and some even grew to layer I (see arrows) at P6. (C, D) At P8, axons in control and mutant mice had grown to the superficial layer of cortex. However, the innervation patterns were different. Controls showed more axon innervation in the lateral S1 with dense callosal innervation at S1/S2 border (C). Mutants showed slightly more axon innervation in the medial S1 (D). (E) The fluorescence density of mutant mice in S1 was significantly higher than in control mice at P6 which suggested that the mutants had increased axon innervation in contralateral S1 at P6. P = 0.003. Scale bar: 500μm for all images. The square brackets in all images outline the S1. The arrow heads in all images outline the S1/S2 border. White lines outline different layers in the cortex of Figure A-D. M: medial; L: lateral.

Article Snippet: Antibodies for intraventricular injection : commercial anti-NMDAR antibody is against amino acid residues 385-399 in the N-terminus of NR1 and was made in Rabbit (AGC-001, Alomone labs).

Techniques: Control, Mutagenesis, Fluorescence

Journal: bioRxiv

Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

doi: 10.1101/2020.06.03.130559

Figure Lengend Snippet: (A) In control mice ( Emx1 cre/+ ; NR1 fl/wt ), cleaved caspase-3 + cells were mostly detected in layer II/III of M1 (A’), only rare cell death was observed in other cortical regions, such as S1 (A’’). (B) Compared with controls, there was increased cell death in layer II/III of motor cortex in mutant mice ( Emx1 cre/+ ; NR1 fl/fl ) (B’). However, compared with controls, there was no increased cell death in other cortical regions in mutant mice, such as S1 (B’’). Scale bar: 500μm for A and B; 200μm for A’, A’’, B’ and B’’.

Article Snippet: Antibodies for intraventricular injection : commercial anti-NMDAR antibody is against amino acid residues 385-399 in the N-terminus of NR1 and was made in Rabbit (AGC-001, Alomone labs).

Techniques: Control, Mutagenesis

Journal: bioRxiv

Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

doi: 10.1101/2020.06.03.130559

Figure Lengend Snippet: (A-D) Deleting NMDAR specifically in projecting neurons. Vectors expressing Cre-recombinase (Cre) and EGFP were delivered into S1 of floxed NR1 mice ( NR1 fl/wt x NR1 fl/wt ) by in utero electroporation at E15.5 (A). Callosal innervation patterns at P14 in control NR1 ipsiS1+/+ mice (B) and NR1 ipsiS1−/− mice (C). (D) Quantification of fluorescence density. P = 0.317. (E-H) Deleting NMDAR specifically in target neurons. NR1 was deleted in target contralateral S1 by in utero electroporation of Cre at E12.5 in NR1 fl/fl ; Ai14 fl/fl mice, the ipsilateral projecting neurons were labeled by EGFP at E15.5 (E). Compared with control NR1 wt/wt ; Ai14 fl/fl (F), NR1 fl/fl ; Ai14 fl/fl mice which specifically deleted NR1 in target S1 showed increased callosal innervation in S1 as “*” shows (G). (H) Quantification of fluorescence density. P = 0.002. Scale bar: 500μm for all images.

Article Snippet: Antibodies for intraventricular injection : commercial anti-NMDAR antibody is against amino acid residues 385-399 in the N-terminus of NR1 and was made in Rabbit (AGC-001, Alomone labs).

Techniques: Expressing, In Utero, Electroporation, Control, Fluorescence, Labeling

Journal: bioRxiv

Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

doi: 10.1101/2020.06.03.130559

Figure Lengend Snippet: (A) NR1 was deleted in target contralateral S1 by in utero electroporation of Cre at E13.5 in NR1 fl/fl ; Ai14 fl/fl mice, the ipsilateral projecting neurons were labeled by EGFP at E15.5. Compared with control NR1 wt/wt ; Ai14 fl/fl (B), NR1 fl/fl ; Ai14 fl/fl , with NR1 specifically deleted in upper cortical layers did not show increased callosal innervation in (C). (D) Quantification of fluorescence density. P = 0.27. Scale bar: 500μm for all images.

Article Snippet: Antibodies for intraventricular injection : commercial anti-NMDAR antibody is against amino acid residues 385-399 in the N-terminus of NR1 and was made in Rabbit (AGC-001, Alomone labs).

Techniques: In Utero, Electroporation, Labeling, Control, Fluorescence

Journal: bioRxiv

Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

doi: 10.1101/2020.06.03.130559

Figure Lengend Snippet: (A) Anti-NR1 antibodies were injected into the lateral ventricle from P2 to P8 and mice were perfused 3 hours later after last injection. Rabbit IgG served as control. Mouse brains then were stained with anti-Rabbit secondary coupled to Alexa594. The red fluorophore of Alexa594 indicated where the antibodies had distributed to. Scale bar: 500μm for all images. (B, B’) In control, the fluorescence signals were mostly detected in the cortex of the ipsilateral injection side, and few in the contralateral cortex. In the ipsilateral injection side, the signals were detected in all the cortical layers, but most strongly in the pia, layer I, layer V, layer VI, cingulum and corpus callosum (see arrows). The signals were also detected in the hippocampus and contralateral motor cortex (see arrows). (C, C’) The general antibody distribution pattern was similar as seen in control. Moreover, the anti-NR1 antibody can bind to NMDAR on the cell membranes, which thus showing beautiful cell membrane staining (see arrows in C’). Scale bar: 500 μm for Figure B, C; 200 μm for Figure B’, C’. CC: corpus callosum; cg: cingulum; Hip: hippocampus; M: motor cortex; S1: primary somatosensory cortex; S2: secondary somatosensory cortex.

Article Snippet: Antibodies for intraventricular injection : commercial anti-NMDAR antibody is against amino acid residues 385-399 in the N-terminus of NR1 and was made in Rabbit (AGC-001, Alomone labs).

Techniques: Injection, Control, Staining, Fluorescence, Membrane

Journal: bioRxiv

Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

doi: 10.1101/2020.06.03.130559

Figure Lengend Snippet: (A-D) Anti-NR1 antibodies were injected into the lateral ventricle from P2 to P12 in ipsilateral cortex. RbIgG served as control. Compared with control (B), antibody injection in mice did not show increased callosal innervation in S1 at P14 (C). (D) Quantification of fluorescence density. P = 0.94. (E-H) Anti-NR1 antibodies were injected into the lateral ventricle from P2 to P12 in contralateral cortex. Compared with control (F), antibody injection in mice showed increased callosal innervation in S1 at P14 (see “*”, G). (H) Quantification of fluorescence density. P =0.0002. Scale bar: 500μm for all images.

Article Snippet: Antibodies for intraventricular injection : commercial anti-NMDAR antibody is against amino acid residues 385-399 in the N-terminus of NR1 and was made in Rabbit (AGC-001, Alomone labs).

Techniques: Injection, Control, Fluorescence

Journal: bioRxiv

Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

doi: 10.1101/2020.06.03.130559

Figure Lengend Snippet: (A-D) Anti-NR1 antibodies were injected into the lateral ventricle from P4 to P8 in contralateral cortex. RbIgG served as control. Compared with control (B), antibody injection in mice show increased callosal innervation in S1 at P14 (C). (D) Quantification of fluorescence density. P = 0.004. (E-H) Anti-NR1 antibodies were injected into the lateral ventricle from P8 to P14 in contralateral cortex. Compared with control (F), antibody injection in mice did not show increased callosal innervation in S1 at P14 (G). (H) Quantification of fluorescence density. P = 0.69. Scale bar: 500μm for all images.

Article Snippet: Antibodies for intraventricular injection : commercial anti-NMDAR antibody is against amino acid residues 385-399 in the N-terminus of NR1 and was made in Rabbit (AGC-001, Alomone labs).

Techniques: Injection, Control, Fluorescence

Journal: bioRxiv

Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

doi: 10.1101/2020.06.03.130559

Figure Lengend Snippet: (A) The callosal innervation pattern in S1 at P30 in control mice ( Emx1 cre/+ ; NR2A fl/wt ) is similar as the pattern in P14 WT control mice, with few axons in S1 but a dense innervation at S1/S2 border. (B) In the mutant mice ( Emx1 cre/+ ; NR2A fl/fl ), the general innervation pattern was as same as control. However, the increased callosal innervation at the border of M1 and S1 was persistent at P30 (see “*” in B’). (C) Quantification of fluorescence density. P = 0.63. (D) In control Emx1 cre/+ ; NR2B fl/wt mice, the callosal innervation pattern at P30 was as normal as WT control. (E) However, the increased callosal innervation in Emx1 cre/+ ; NR2B fl/fl mice lasted at least to P30 as we observed in Emx1 cre/+ ; NR1 fl/fl mice at P30. (F) Quantification of fluorescence density. P = 0.007. Scale bar: 500μm for all images.

Article Snippet: Antibodies for intraventricular injection : commercial anti-NMDAR antibody is against amino acid residues 385-399 in the N-terminus of NR1 and was made in Rabbit (AGC-001, Alomone labs).

Techniques: Control, Mutagenesis, Fluorescence

Journal: bioRxiv

Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

doi: 10.1101/2020.06.03.130559

Figure Lengend Snippet: (A, B) EPHB2 protein expression are decreased in Emx1 cre/+ ; NR1 fl/fl mice at P5. In control Emx1 cre/+ ; NR1 wt/wt mice, EPHB2 was expressed both in CC and cortex (A). EPHB2 in Emx1 cre/+ ; NR1 fl/fl mice was dramatically decreased in cortex (B). (C) Western blot analysis of cortical protein extracts from P8 S1 showed that, relative to the loading control beta-tubulin (β-Tub) and GAPDH, lower levels of EPHB2 were observed in the five samples of Emx1 cre/+ ; NR1 fl/fl mice compared to the five samples of controls. (D) Quantification of protein levels relative to β-Tub. P=0.001. (E) Quantification of protein levels relative to GAPDH. P< 0.0001. (F) The quantitative polymerase chain reaction (qPCR) analysis showed no expression difference of EPHB2 between Emx1 cre/+ ; NR1 fl/fl mice and controls. Scale bar: 500μm for A, B.

Article Snippet: Antibodies for intraventricular injection : commercial anti-NMDAR antibody is against amino acid residues 385-399 in the N-terminus of NR1 and was made in Rabbit (AGC-001, Alomone labs).

Techniques: Expressing, Control, Western Blot, Real-time Polymerase Chain Reaction

Journal: Annals of neurology

Article Title: Absence of Neuronal Autoantibodies in Neuropsychiatric Systemic Lupus Erythematosus.

doi: 10.1002/ana.25908

Figure Lengend Snippet: Figure 1. Clinical features and immunoglobulin G (IgG) binding to conformational neuronal surface epitopes from plasma and cerebrospinal fluid (CSF) of patients with neuropsychiatric lupus (NPSLE). A. Clinical features of 35 patients with SLE, including 15 with active NPSLE at the time of sampling. 32/34 (94%) were on immunotherapies, including prednisolone (71%), hydroxychloroquine (41%), belimumab (6%) and rituximab (3%). ANA = anti-nuclear antibody. B. Live cell-based assays employing HEK293T cells with surface expressed N-methyl D-aspartate receptors (NMDARs) as NR1-NR2A or NR1-NR2B heteromers (nuclei highlighted with DAPI, 4′,6-diamidino-2-phenylindole). First panel shows that a commercial antibody directed against the extracellular domain of the NR2A-subunit (red) binds to HEK293T cells which express NR1-NR2A heteromers. NMDAR-antibody encephalitis (NMDAR-Ab-E) patient serum IgGs (n=11) bind to the NR1-NR2A heteromers (second panel), and to NR1-NR2B heteromers / NR1 homomers (data not shown). NMDAR-antibody patient CSFs show similar binding (as Irani et al, 2010).10 Serum from healthy controls (n=36), plasma and CSFs from SLE patients (n=35 of each) show no binding. No binding was observed with the G11 antibody (data not shown, lower panel representative). Throughout, IgG binding was visualized with an anti-human 568-Alexaflour antibody (1:750 dilution, A-21090) C. Commercial antibodies against the extracellular domains of NR1 (Alomone, AGC-001), NR2A (Alomone,

Article Snippet: Throughout, IgG binding was visualized with an anti-human 568-Alexaflour antibody (1:750 dilution, A-21090) C. Commercial antibodies against the extracellular domains of NR1 (Alomone, AGC-001), NR2A (Alomone, AGC-002) and NR2B (kind gift from Prof Stephenson, London) subunits bound to the surface of live HEK293T cells co-transfected with EGFP plus NR1 and NR2B subunits.

Techniques: Binding Assay, Clinical Proteomics, Sampling

Journal: Frontiers in Neuroscience

Article Title: Altered Light Sensitivity of Circadian Clock in Shank3 +/– Mouse

doi: 10.3389/fnins.2021.604165

Figure Lengend Snippet: Immunostaining for cholera toxin β-subunit (CTB) and NMDAR2A in the suprachiasmatic nucleus (SCN) from wild-type (WT) and Shank3 +/– mice. CTB (diaminobenzidine, DAB, left column ) and NMDAR2A ( right column ) did not show obvious differences in the immunoreactivity between groups (WT, top micrographs ; Shank3 +/– , bottom micrographs ). For this and subsequent figures ( , ), scale bar = 50 μm.

Article Snippet: Primary antibodies were diluted as indicated in 0.1 M PBS containing 1.0% normal serum in 0.3% Triton X-100 [anti-VIP raised in rabbit, CAT 20077, Incstar, 1:2,000 ( ); anti-CTB subunit raised in goat, CAT 703, List Biological Laboratories, 1:2,000 ( ); anti-NMDAR2A (GluN2A) raised in rabbit, CAT ACG 002, Alomone Labs, 1:400 ( ); and anti c-FOS raised in rabbit, CAT SC-52, Santa Cruz, 1:1,000 ( )].

Techniques: Immunostaining

Journal: International Journal of Molecular Sciences

Article Title: AMPA and NMDA Receptors in Hippocampus of Rats with Fluoride-Induced Cognitive Decline

doi: 10.3390/ijms252111796

Figure Lengend Snippet: mRNA and protein levels of GluN1 subunits of NMDARs in hippocampal cells. ( A ) Relative expression of Grin1 gene in the hippocampus of rats consumed different F - doses normalized to that of a pair of reference genes ( Eef1a+Ppia ). Average values ± SEM (n = 10). ( B , C ) Protein expression of native and phosphorylated (Ser890) forms of GluN1 subunit in cytosolic and membrane fractions, respectively. Shown are typical immunoblots and average values ± SEM (n = 8).

Article Snippet: Rabbit antibodies to GluA1 (#13185), phospho-GluA1 (#8084), GluA2 (#5306), phospho-GluA2 (#3921), GluA3 (#4676), GluN1 (#5704), phospho-GluN1 (#3381), GluN2A (#4205), phospho-GluN2A (#4206), GluN2B (#4207) and phospho-GluN2B (#4208) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Membrane, Western Blot

Journal: bioRxiv

Article Title: A Consequence of Immature Breathing induces Persistent Changes in Hippocampal Synaptic Plasticity and Behavior: A Role of Pro-Oxidant State and NMDA Receptor Imbalance

doi: 10.1101/2023.03.21.533692

Figure Lengend Snippet: A . ( left ) Representative traces of the evoked fEPSP from control (black), control+AP5 [50µM] (green), nIH (red) and nIH +AP5 [50µM] (blue) in baseline conditions prior to TBS (1) and following TBS (2). ( middle ) Mean fEPSP slope plotted as a function of time relative to the slope before TBS in control, control+AP5, nIH and nIH+AP5. ( right ) fEPSP slope represented as percent change from baseline at 60 min after TBS in control slices vs control+AP5 [50 µM]. B . ( top ) Representative image for GluN1. (bottom) The graph shows GluN1 did not change protein expression after nIH exposure compared with control. (two tailed t-test, t=0.63; df=4.1; P=0.55). C . (top) Western blot picture for GluN2A. (bottom) Comparison for both conditions show GluN2A decreased the protein content levels after nIH. (two tailed t-test, t=4.017; df=6.43; P=0.006). D . ( top ) Representative immunoblot image for GluN2B. (bottom) Comparison of both conditions show increased GluN2B levels after nIH (two tailed t-test, t=3.43; df=5.78; P=0.014). E . GluN2B/GluN2A comparison ratio. F . ( left ) Representative traces of the evoked fEPSP control +TCN [5 µM] (orange), control + ifenprodil [5 µM] (cyan) and control + both drugs (olive) in baseline conditions prior to TBS (1) and following TBS (2). ( middle ) Mean fEPSP slope plotted as a function of time relative to the slope before TBS and ( right ) Comparison of fEPSP slope represented as percent change from baseline at 60 min after TBS (one way ANOVA, F (3,20) =43.52; P<0.001). Black dashed line represents the mean slope of the fEPSP 60 min following TBS in control slices from . G . ( left ) Representative traces of the evoked fEPSP from nIH+TCN [5 µM] (dark yellow) and nIH +ifenprodil [5 µM] (light blue) in baseline conditions prior to TBS (1) and following TBS (2). ( middle ) Mean fEPSP slope plotted as a function of time and relative to slope before TBS ( right ) fEPSP slope represented as percent change from baseline at 60 min after TBS (two tailed t-test, t=12.46; df=4.59; P=0.001). Red dashed line represents the mean slope of the fEPSP 60 min following TBS in nIH slices from . Scale bars for A, F and G: 10 msec x 0.2 mV. The box-plot parameters indicate mean ± S.E. The analysis was performed for A to E using unpaired two-tailed t-test with Welch’s correction and for F and G the analysis was performed using one-way ANOVA followed by Bonferroni post hoc. **P<0.01, ***P<0.001 and ****P<0.0001).

Article Snippet: Membranes were incubated under constant shaking with primary antibodies: anti rabbit GluN1 (1:2000; Abcam Cat# ab109182, RRID:AB_10862307) anti-rabbit GluN2A (1:2000; Cell Signaling Technology Cat# 4205, RRID:AB_2112295), anti-rabbit GluN2B (1:2000; Cell Signaling Technology Cat# 14544, RRID:AB_2798506), anti-rabbit NOX2 (1:2000; Abcam Cat# ab129068, RRID:AB_11144496) anti-rabbit NOX4 (1:500; Novus Cat# NB110-58851B, RRID:AB_1217375 and anti-mouse GAPDH (1: 10.000; Abcam Cat# ab8245, RRID:AB_2107448).

Techniques: Control, Expressing, Two Tailed Test, Western Blot, Comparison

Journal: bioRxiv

Article Title: A Consequence of Immature Breathing induces Persistent Changes in Hippocampal Synaptic Plasticity and Behavior: A Role of Pro-Oxidant State and NMDA Receptor Imbalance

doi: 10.1101/2023.03.21.533692

Figure Lengend Snippet: A . Malondialdehyde (MDA) content was measured in hippocampal homogenates from control, nIH Saline and 10-Mn. (one way ANOVA, F (2,12) =11.53; P=0.0016). B . (top) Representative blot of nuclear HIF1a performed from control, nIH Saline and IH 10-Mn mice. (bottom) Quantification of HIF1a expression (one way ANOVA, F (2,12) =5.76; P=0.017). C . (top) Immunoblot of NOX2. (bottom) Significant differences was found in hippocampal homogenate from nIH Saline compared to control and nIH Mn (one way ANOVA, F (2,15) =9.26; P=0.0024). D . (top) Representative image of NOX4. ( bottom ) Comparison of NOX4 expression between control, nIH Saline and nIH Mn . (one way ANOVA, F (2,9) =11.46; P=0.0034). E . ( left ) Representative blot of GluN2A from control, nIH Saline and nIH Mn mice. ( right ). Quantification of GluN2A expression. (one way ANOVA, F (2,15) =6.63; P=0.0086). F . ( left ) Immunoblot of GluN2B. ( right ) Significant differences were found in hippocampal homogenate from nIH Saline vs control and nIH Mn . (one way ANOVA, F (2,12) =6.88; P=0.01). G . (top) Representative traces of evoked fEPSP from nIH Mn (purple) in baseline conditions prior to TBS (1) and following TBS (2). ( left bottom ) Mean fEPSP slope plotted as a function of time relative to the slope before TBS in nIH Mn . ( right ) fEPSP slope represented as percent change from baseline at 60 min after TBS in nIH vs nIH Mn slices. (two tailed t-test, t=3.41; df=10.61; P=0.0061). Dashed lines represent the mean slope of the fEPSP 60 min following TBS in control (black dashed line) and nIH (red dashed line) slices from . H . (top) Representative traces of the evoked fEPSP from nIH Mn in presence the ifenprodil [5 µM] (light purple) in baseline conditions prior to TBS (1) and following TBS (2). ( left bottom ) Mean fEPSP slope plotted as a function of time relative to the slope before TBS in nIH Mn in presence the ifenprodil. ( right ) fEPSP slope represented as percent change from baseline at 60 min after TBS in IH+ifenprodil vs nIH Mn in presence of ifenprodil. (two tailed t-test, t=3.99; df=6.99; P=0.019). Blue dashed line represents the mean slope of the fEPSP 60 min following TBS in ifenprodil treated nIH slices from . For G and H, Scale bars 10 msec x 0.2 mV. The analysis was performed for A to F using one-way ANOVA followed by Bonferroni post hoc and for G and F, the analysis was performed using unpaired two-tailed t-test with Welch’s correction. *P<0.05, **P<0.01, and ***P<0.001.

Article Snippet: Membranes were incubated under constant shaking with primary antibodies: anti rabbit GluN1 (1:2000; Abcam Cat# ab109182, RRID:AB_10862307) anti-rabbit GluN2A (1:2000; Cell Signaling Technology Cat# 4205, RRID:AB_2112295), anti-rabbit GluN2B (1:2000; Cell Signaling Technology Cat# 14544, RRID:AB_2798506), anti-rabbit NOX2 (1:2000; Abcam Cat# ab129068, RRID:AB_11144496) anti-rabbit NOX4 (1:500; Novus Cat# NB110-58851B, RRID:AB_1217375 and anti-mouse GAPDH (1: 10.000; Abcam Cat# ab8245, RRID:AB_2107448).

Techniques: Control, Saline, Expressing, Western Blot, Comparison, Two Tailed Test

Journal: bioRxiv

Article Title: A Consequence of Immature Breathing induces Persistent Changes in Hippocampal Synaptic Plasticity and Behavior: A Role of Pro-Oxidant State and NMDA Receptor Imbalance

doi: 10.1101/2023.03.21.533692

Figure Lengend Snippet: A . (top) Representative traces of the evoked fEPSP from adult control (black) and adult mice were exposed to neonatal IH (red) in baseline conditions prior to TBS (1) and following TBS (2). ( left bottom ) Mean fEPSP slope plotted as a function of time and relative to baseline prior to TBS. ( right bottom ) fEPSP slope represented as percent change from baseline at 60 min following TBS in adult control vs Adult nIH (two tailed t-test, t=5.70; df=9.04; P=0.003). B . (top) Representative trace of the evoked from control+ TCN-213 [5 µM] and control + ifenprodil [5 µM] in baseline conditions prior to TBS (1) and following TBS (2). ( left bottom ) Mean fEPSP slope plotted as a function of time and relative to baseline prior to TBS. ( right bottom ) fEPSP comparison at 60 min following TBS in control +TCN and control+ ifenprodil (two tailed t-test, t=7.43; df=4.65; P=0.0009). Black dashed line represents the mean slope of the fEPSP 60 min following TBS in control slices from . C . (top) Representative trace of the evoked response from Adult nIH + TCN-213 [5 µM] and Adult nIH + ifenprodil [5 µM] in baseline conditions prior to TBS (1) and following TBS (2). ( left bottom ) Mean fEPSP slope plotted as a function of time relative to baseline prior to TBS. ( right bottom ) fEPSP slope represented as percent change from baseline at 60 min following TBS (two tailed t-test, t=4.72; df=4.06; P=0.0064). Red dashed line represents the mean slope of the fEPSP after 60 min following TBS in control slices from . D . (top) Representative traces of the evoked fEPSP from adult mice was exposure to neonatal IH+10 days of MnTMPyP (Adult nIH-Mn ) and adult mice receive MnTMPyP after exposure to neonatal IH (Adult REC-Mn ) in baseline conditions before TBS (1) and after TBS (2). ( left bottom ) Mean fEPSP slope plotted as a function of time and relative to slope before TBS in Adult nIH-Mn vs Adult REC-Mn ( right bottom ) fEPSP slope represented as percent change from baseline at 60 min after TBS Adult nIH-Mn vs Adult REC-Mn (two tailed t-test, t=3.79; df=8.16; P=0.0051). E. Representative image of GluN1. ( bottom ) Quantification shows GluN1 protein expression is not changed in Adult nIH , Adult nIH-Mn or 10+Mn exposures compared to control (one way ANOVA, F (3,16)=1.14 ; P=0.93; N=5). F . (top) Representative blot of GluN2A performed from adult mice unexposed, Adult nIH , Adult nIH-Mn or Adult REC-Mn . ( bottom ). Quantification of GluN2A expression from adult control, Adult nIH , Adult nIH-Mn or 10+Mn. (one way ANOVA, F (3,12)=8.31 ; P=0.0029, N=4). G . (top) Immunoblot of GluN2B. ( bottom ) Significant differences were found in hippocampal homogenates from adult control, Adult nIH , Adult nIH-Mn or Adult REC-Mn (one way ANOVA, F (3,12)=4.91 ; P=0.018, N=4). The box plot parameters indicate mean ± S.E. The analysis was performed for A-D using unpaired two-tailed t-test with Welch’s correction. The analysis was performed for E-G using one-way ANOVA followed by Bonferroni post hoc. *P=0.05, **P=0.01, ***P=0.001 and N.S= no significant. Scale bars for A,B, F and G= 10 msec x 0.2 mV

Article Snippet: Membranes were incubated under constant shaking with primary antibodies: anti rabbit GluN1 (1:2000; Abcam Cat# ab109182, RRID:AB_10862307) anti-rabbit GluN2A (1:2000; Cell Signaling Technology Cat# 4205, RRID:AB_2112295), anti-rabbit GluN2B (1:2000; Cell Signaling Technology Cat# 14544, RRID:AB_2798506), anti-rabbit NOX2 (1:2000; Abcam Cat# ab129068, RRID:AB_11144496) anti-rabbit NOX4 (1:500; Novus Cat# NB110-58851B, RRID:AB_1217375 and anti-mouse GAPDH (1: 10.000; Abcam Cat# ab8245, RRID:AB_2107448).

Techniques: Control, Two Tailed Test, Comparison, Expressing, Western Blot

Journal: Neurobiology of disease

Article Title: Ethanol exposure alters Alzheimer’s-related pathology, behavior, and metabolism in APP/PS1 mice

doi: 10.1016/j.nbd.2022.105967

Figure Lengend Snippet: Chronic moderate drinking differentially alters NMDA and GABAA receptors in the cortex and hippocampus of APP/PS1 mice. a) Ethanol treatment did not alter cortical Grin2a expression in wildtype or APP/PS1 mice. b) Ethanol-treated APP/PS1 mice had higher cortical Grin2b expression compared to EtOH-treated wildtype mice. 2-way ANOVA revealed a significant treatment × genotype interaction ( p = 0.0319). c) H 2 O-treated APP/PS1 mice showed increased cortical Gabra5 expression compared to H 2 O-exposed wildtype mice (p < 0.05). This effect was lost in EtOH-exposed APP/PS1 Gabra5 mRNA levels. 2-way ANOVA revealed a significant treatment × genotype interaction ( p = 0.0249) and a trend in genotype effects ( p = 0.0723). d) Synaptic GluN2A levels was unaltered in the hippocampus of H 2 O- or EtOH-treated wildtype or APP/PS1 mice. e) Synaptic GluN2B levels was unaltered in the hippocampus of H 2 O- or EtOH-treated wildtype or APP/PS1 mice. f) Ethanol-treated wildtype mice showed increased synaptic GABA A R α5 subunit levels compared to H 2 O-treated wildtype mice. Ethanol treatment had no effect on GABAAR α5 subunit levels in APP/PS1 mice. 2-way ANOVA revealed a significant treatment × genotype effect ( p = 0.0347) and a trend in treatment effects ( p = 0.0644). Wildtype + H2O, n = 10; APP/PS1 + H2O, n = 9; Wildtype + EtOH, n = 7; APP/PS1 + EtOH, n = 8. * p < 0.05.

Article Snippet: The following primary and secondary antibodies were used for this study: APP (including CTFβ and CTFα; Invitrogen; CT695; 1:1000), BACE1 (Cell Signaling; 5606S; 1:1000), ADAM10 (Millipore; AB19026; 1:1000), IDE (Abcam; ab232216; 1:1000), GluN2A (Cell Signaling; 4025; 1:1000), GluN2B (Cell Signaling; 4212; 1:1000), GABAAR α5 (Santa Cruz; Sc393921; 1:1000), and β-actin (Millipore; MAB1501; 1:50,000), anti-mouse (Cell Signaling; 7076S; 1:5000), anti-rabbit (Cell Signaling; 7074S; 1:5000).

Techniques: Expressing

Journal: Journal of Affective Disorders Reports

Article Title: Long-term cyclosporine A treatment promotes anxiety-like behavior: Possible relation with glutamate signaling in rat hippocampus

doi: 10.1016/j.jadr.2022.100394

Figure Lengend Snippet: Fig. 4. Western blot analysis of NMDA receptors in the hippocampus of control and CsA-treated rats. (A) Representative images of the levels of total (pan) GluN2, GluN2A, GluN2B, p-GluN1 (Ser897) and total GluN1. (B-F) Respective quantifications of immunodetections normalized to β-actin and presented as a percentage of the control mean. Data represent the mean ± SEM. Statistics: ** *p ≤0.001 as determined by Student’s t-test; n = 6.

Article Snippet: The utilized primary antibodies and respective dilutions were as follows: anti-AMPAR (1:500, #13185 Cell Signaling Technology), phosphorylated AMPAR (pAMPAR-S831) (1:1000, A4352-Sigma–Aldrich), pAMPAR (S845) (1:1000, #8084 – Cell Signaling Technology), GluN1 (1:750, #G8913 – Sigma–Aldrich), pan-GluN2 (1:750, 244-0P – SYSY Synaptic Systems), GluN2A (1:1000, #4205 - Cell Signaling Technology), GluN2B (1:1000, #4207 - Cell Signaling Technology), pGluN1 (Ser897) (1:500, 3385S – Cell Signaling Technology), nNOS (1:500, SC648 – Santa Cruz Biotechnology), and iNOS (1:750, SC7271 – Santa Cruz Biotechnology). β-actin antibody (1:5000, A5441 – Sigma-Aldrich) was used as a loading control in the experiments.

Techniques: Western Blot, Control