Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Single-molecule imaging of the functional crosstalk between surface NMDA and dopamine D1 receptors

doi: 10.1073/pnas.1310145110

Figure Lengend Snippet: The D1R–NMDAR interaction bidirectionally regulates the surface distribution and dynamics of D1R and NMDAR. (A) Immunostaining of surface D1R-CFP (green) and GluN1 subunit (red) in hippocampal neurons. The yellow arrow shows overlay. (B) Immunostaining of surface D1R-CFP in control or after D1/5R agonist, TAT-t2, or TAT-[N2A15] application. (Scale bar, 250 nm.) (C) Normalized measures of D1R-CFP clusters intensity in control (n = 32 neuronal fields), D1/5R agonist-treated (n = 24 neuronal fields; *P < 0.05 compared with control), TAT-NSt2–treated (non-sense of TAT-t2, n = 19 neuronal fields), TAT-t2–treated (n = 21 neuronal fields; **P < 0.01 compared with TAT-NSt2), TAT-NSt3–treated (non-sense of TAT-t3, n = 11 neuronal fields), TAT-t3–treated (n = 12 neuronal fields; P > 0.05 compared with TAT-NSt3), TAT-[NS15]–treated (n = 27 neuronal fields; P > 0.05), or TAT-[N2A15]–treated (n = 21 neuronal fields, *P < 0.05 compared with TAT-[NS15]) conditions. (D) Representative trajectories (1,000 frames, 20-Hz acquisition rate) of surface single D1R-CFP (Left) (green) (scale bar, 400 nm) and GluN1-NMDAR (Right) (blue) (scale bar, 300 nm) in the absence and presence of either D1/5R agonist (10 µM, 15 min) or TAT-t2 (10 µM, 15 min). Bold dotted line, perisynaptic area; thin dotted line, PSD area. (E) Plot of the MSD of surface D1R-CFP (Upper) (green) and GluN1-NMDAR (Lower) (blue) versus time in presence of TAT-NS or TAT-t2 peptides (10 µM, 15 min). The SEM is included for each data point (D1R: TAT-NS, n = 986 trajectories, and TAT-t2, n = 1,326; GluN1-NMDAR: TAT-NS, n = 198, and TAT-t2, n = 134). (F and G) Representative surface distributions of single D1R-CFP (green) (F) and GluN1-NMDAR (blue) (G) in the synaptic area (PSD + perisynaptic area) in control, D1/5R agonist, and TAT-t2 conditions. Each dot represents the detection of a single receptor during a frame. Comparisons of the time spent in the synaptic area (dwell time) by single D1R-CFP (control, n = 173 trajectories; D1/5R agonist, n = 142, **P < 0.01; TAT-t2, n = 752, *P < 0.05) (F) and GluN1-NMDAR (control, n = 189 trajectories; D1/5R agonist, n = 157, *P < 0.05; TAT-t2, n = 134, **P < 0.01) (G) and the synaptic fraction of detected single D1R-CFP (control, n = 14 neuronal fields; D1/5R agonist, n = 19, **P < 0.01; D1/5R agonist in the presence of dynasore, n = 47, **P < 0.01; TAT-t2, n = 15, ***P < 0.001) (F), D5R-CFP (n = 16, P > 0.05) (F), and GluN1-NMDAR (control, n = 11; D1/5R agonist, n = 15, *P < 0.05; TAT-t2, n = 14, *P < 0.05) (G). Dyn., dynasore; D1/5 ago., D1/5 receptor agonist SKF-38393.

Article Snippet: For single-nanoparticle tracking, QD 655 coupled to goat anti-rabbit F(ab′) 2 or anti-mouse IgG (Invitrogen) was incubated (1:10,000, 10 min) onto neurons previously exposed for 10 min to either mouse monoclonal anti-GFP (1 µg; Invitrogen), rabbit polyclonal anti-D1R (1 µg; Lifespan Biosciences), mouse monoclonal anti-GluA2:00 AMPAR subunit (1 µg; Millipore), or rabbit polyclonal anti–GluN1-NMDAR subunit (1 µg; Alomone Laboratories) antibodies.

Techniques: Immunostaining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Single-molecule imaging of the functional crosstalk between surface NMDA and dopamine D1 receptors

doi: 10.1073/pnas.1310145110

Figure Lengend Snippet: D1R activation or D1R/GluN1-NMDAR interaction blockade increases synaptic NMDAR content and favors AMPAR synaptic long-term potentiation. (A) (Left) Excitatory postsynaptic current traces recorded at −70 mV and +40 mV from a representative hippocampal CA1 pyramidal cell, before and 10 min after exposure to D1/5R agonist. (Right) Relative change over time of the AMPA/NMDA ratio at CA1 synapses in the absence or presence of D1/5R agonist (n = 13, *P < 0.05 10 min after agonist) and in the absence or presence of vehicle (n = 7, P > 0.05). (B) Surface imaging of GluN1-SEP in neurons incubated with either TAT-NS or TAT-t2 (10 µM). (Scale bar, 5 µm.) (Right) Average value of GluN1-SEP content in the synaptic area after TAT-NS or TAT-t2 application (n = 8 neurons per group, **P < 0.01). (C) Dendritic fragment of a hippocampal neuron expressing Homer 1c-DsRed (Upper) and GluA1-SEP (Lower). SEP only fluoresces at neutral pH when receptors are inserted at the plasma membrane. Ten minutes after chemical LTP induction (cLTP), the GluA1-SEP fluorescence intensity increased in postsynaptic clusters. (Insets) High magnification of a synaptic GluA1-SEP cluster. (Scale bar, 2 µm.) (D) Comparison of the synaptic GluA1-SEP fluorescence intensity before and after cLTP with prior TAT-NS (n = 198 synapses, *P < 0.05) or TAT-t2 (n = 215 synapses, *P < 0.05) (TAT-NS versus TAT-t2; *P < 0.05) application. (E) Schematic model of the D1R–NMDAR surface interplay in hippocampal neurons. D1Rs are highly diffusive at the neuronal surface and are dynamically retained in clusters in the vicinity of glutamate synapses where they interact with NMDAR. Dopamine release disrupts this interaction and favors the lateral redistribution of both receptors: D1Rs freely explore extrasynaptic areas, whereas NMDARs laterally reach the PSD where they impact on the long-term plasticity of glutamate synapses.

Article Snippet: For single-nanoparticle tracking, QD 655 coupled to goat anti-rabbit F(ab′) 2 or anti-mouse IgG (Invitrogen) was incubated (1:10,000, 10 min) onto neurons previously exposed for 10 min to either mouse monoclonal anti-GFP (1 µg; Invitrogen), rabbit polyclonal anti-D1R (1 µg; Lifespan Biosciences), mouse monoclonal anti-GluA2:00 AMPAR subunit (1 µg; Millipore), or rabbit polyclonal anti–GluN1-NMDAR subunit (1 µg; Alomone Laboratories) antibodies.

Techniques: Activation Assay, Imaging, Incubation, Expressing, Fluorescence

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: Neural Regeneration Research

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

doi: 10.4103/1673-5374.184494

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

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

Techniques: Expressing, Western Blot, Control, Injection

Journal: Neural Regeneration Research

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

doi: 10.4103/1673-5374.184494

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

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

Techniques: Control, Injection, Immunostaining, Membrane

Journal: 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, 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:

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: 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: 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, 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, 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, Staining, 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 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, 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, 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: 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, Western Blot, Real-time Polymerase Chain Reaction