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    Structured Review

    Alomone Labs glun1
    No increased cell death in S1 of Emx1 cre/+ ; <t>Grin1</t> fl/fl mice at P6. ( A ) In control mice ( Emx1 cre/+ ; Grin1 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/+ ; Grin1 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: 500 μm for A and B ; 200 μm for A’ , A’’ , B’ , and B’’ .
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    Images

    1) Product Images from "NMDA receptors control development of somatosensory callosal axonal projections"

    Article Title: NMDA receptors control development of somatosensory callosal axonal projections

    Journal: eLife

    doi: 10.7554/eLife.59612

    No increased cell death in S1 of Emx1 cre/+ ; Grin1 fl/fl mice at P6. ( A ) In control mice ( Emx1 cre/+ ; Grin1 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/+ ; Grin1 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: 500 μm for A and B ; 200 μm for A’ , A’’ , B’ , and B’’ .
    Figure Legend Snippet: No increased cell death in S1 of Emx1 cre/+ ; Grin1 fl/fl mice at P6. ( A ) In control mice ( Emx1 cre/+ ; Grin1 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/+ ; Grin1 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: 500 μm for A and B ; 200 μm for A’ , A’’ , B’ , and B’’ .

    Techniques Used: Mouse Assay, Mutagenesis

    The expression of NMDAR in cortex was reduced in Emx1 cre/+ ; Grin1 fl/fl mice. Examples of 12 µm coronal brain sections from P8 Emx1 cre/+ ; Grin1 wt/wt ( A ) and Emx1 cre/+ ; Grin1 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/+ ; Grin1 wt/wt mice revealed a clear barrel pattern ( Aa ). However, the barrel pattern in Emx1 cre/+ ; Grin1 fl/fl mice was disrupted and less distinct ( Ba ). The GluN1 staining in Emx1 cre/+ ; Grin1 wt/wt mice were dense and strong in cortex ( Ab , Ac ). However, the staining in Emx1 cre/+ ; Grin1 fl/fl mice was less bright and apparently reduced in Layer V and VI ( Bb , Bc ). ( C ) Western blot of cortical protein extracts from P8 S1. Relative to the loading control GAPDH, the protein levels of GluN1 were greatly reduced in the four samples of Emx1 cre/+ ; Grin1 fl/fl mice compared to the four samples of controls. ( D ) Quantification of protein levels of GluN1 relative to GAPDH. p=0.0002. Scale bar: 100 μm for Ac and Bc ; 500 μm for rest of images.
    Figure Legend Snippet: The expression of NMDAR in cortex was reduced in Emx1 cre/+ ; Grin1 fl/fl mice. Examples of 12 µm coronal brain sections from P8 Emx1 cre/+ ; Grin1 wt/wt ( A ) and Emx1 cre/+ ; Grin1 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/+ ; Grin1 wt/wt mice revealed a clear barrel pattern ( Aa ). However, the barrel pattern in Emx1 cre/+ ; Grin1 fl/fl mice was disrupted and less distinct ( Ba ). The GluN1 staining in Emx1 cre/+ ; Grin1 wt/wt mice were dense and strong in cortex ( Ab , Ac ). However, the staining in Emx1 cre/+ ; Grin1 fl/fl mice was less bright and apparently reduced in Layer V and VI ( Bb , Bc ). ( C ) Western blot of cortical protein extracts from P8 S1. Relative to the loading control GAPDH, the protein levels of GluN1 were greatly reduced in the four samples of Emx1 cre/+ ; Grin1 fl/fl mice compared to the four samples of controls. ( D ) Quantification of protein levels of GluN1 relative to GAPDH. p=0.0002. Scale bar: 100 μm for Ac and Bc ; 500 μm for rest of images.

    Techniques Used: Expressing, Mouse Assay, Immunostaining, Staining, Western Blot

    NR2B ( Emx1 cre/+ ; Grin2b fl/fl ) but not NR2A ( Emx1 cre/+ ; Grin2a fl/fl ) mice had same disrupted callosal innervation patterns as Emx1 cre/+ ; Grin1 fl/fl at P30. ( A ) The callosal innervation pattern in S1 at P30 in control mice ( Emx1 cre/+ ; Grin2a 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/+ ; Grin2a 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/+ ; Grin2b fl/wt mice, the callosal innervation pattern at P30 was as normal as WT control. ( E ) However, the increased callosal innervation in Emx1 cre/+ ; Grin2b fl/fl mice lasted at least to P30 as we observed in Emx1 cre/+ ; Grin1 fl/fl mice at P30. ( F ) Quantification of fluorescence density. p=0.007. Scale bar: 500 μm for all images.
    Figure Legend Snippet: NR2B ( Emx1 cre/+ ; Grin2b fl/fl ) but not NR2A ( Emx1 cre/+ ; Grin2a fl/fl ) mice had same disrupted callosal innervation patterns as Emx1 cre/+ ; Grin1 fl/fl at P30. ( A ) The callosal innervation pattern in S1 at P30 in control mice ( Emx1 cre/+ ; Grin2a 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/+ ; Grin2a 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/+ ; Grin2b fl/wt mice, the callosal innervation pattern at P30 was as normal as WT control. ( E ) However, the increased callosal innervation in Emx1 cre/+ ; Grin2b fl/fl mice lasted at least to P30 as we observed in Emx1 cre/+ ; Grin1 fl/fl mice at P30. ( F ) Quantification of fluorescence density. p=0.007. Scale bar: 500 μm for all images.

    Techniques Used: Mouse Assay, Mutagenesis, Fluorescence

    Contralateral injection of anti-NMDAR antibodies from P4 to P8 but not P8 to P14 had increased callosal innervation in S1. ( A–D ) Anti-GluN1 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-GluN1 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: 500 μm for all images.
    Figure Legend Snippet: Contralateral injection of anti-NMDAR antibodies from P4 to P8 but not P8 to P14 had increased callosal innervation in S1. ( A–D ) Anti-GluN1 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-GluN1 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: 500 μm for all images.

    Techniques Used: Injection, Mouse Assay, Fluorescence

    There was no difference between Emx1 cre/+ ; Grin1 fl/wt and Emx1 cre/+ ; Grin1 fl/fl mice during axonal extension into the ipsilateral CC ( P0 ) and to the contralateral CC ( P5 ). ( A, B ) The callosal axons in S1 formed a bundle and grew into the ipsilateral CC at P0 in control and GluN1 KO littermates ( Emx1 cre/+ ; Grin1 fl/wt and Emx1 cre/+ ; Grin1 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: 500 μm for all images.
    Figure Legend Snippet: There was no difference between Emx1 cre/+ ; Grin1 fl/wt and Emx1 cre/+ ; Grin1 fl/fl mice during axonal extension into the ipsilateral CC ( P0 ) and to the contralateral CC ( P5 ). ( A, B ) The callosal axons in S1 formed a bundle and grew into the ipsilateral CC at P0 in control and GluN1 KO littermates ( Emx1 cre/+ ; Grin1 fl/wt and Emx1 cre/+ ; Grin1 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: 500 μm for all images.

    Techniques Used: Mouse Assay

    Deleting NMDAR specifically in target neurons. GluN1 was deleted in target contralateral S1 by in utero electroporation of Cre at E12.5 in Grin1 fl/fl ; Rosa26 fs-tdTomato mice, the ipsilateral projecting neurons were labeled by EGFP at E15.5. This figure shows images from all of the animals that were analyzed for the statistical analysis. Scale bar: 500 μm for all images. R26 tdT : Rosa26 fs-tdTomato .
    Figure Legend Snippet: Deleting NMDAR specifically in target neurons. GluN1 was deleted in target contralateral S1 by in utero electroporation of Cre at E12.5 in Grin1 fl/fl ; Rosa26 fs-tdTomato mice, the ipsilateral projecting neurons were labeled by EGFP at E15.5. This figure shows images from all of the animals that were analyzed for the statistical analysis. Scale bar: 500 μm for all images. R26 tdT : Rosa26 fs-tdTomato .

    Techniques Used: In Utero, Electroporation, Mouse Assay, Labeling

    The protein but not RNA level of EPHB2 was reduced in Emx1 cre/+ ; Grin1 fl/fl mice at P8. ( A, B ) EPHB2 protein expression is decreased in Emx1 cre/+ ; Grin1 fl/fl mice at P5. In control Emx1 cre/+ ; Grin1 wt/wt mice, EPHB2 was expressed both in CC and cortex ( A ). EPHB2 in Emx1 cre/+ ; Grin1 fl/fl mice was 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/+ ; Grin1 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
    Figure Legend Snippet: The protein but not RNA level of EPHB2 was reduced in Emx1 cre/+ ; Grin1 fl/fl mice at P8. ( A, B ) EPHB2 protein expression is decreased in Emx1 cre/+ ; Grin1 fl/fl mice at P5. In control Emx1 cre/+ ; Grin1 wt/wt mice, EPHB2 was expressed both in CC and cortex ( A ). EPHB2 in Emx1 cre/+ ; Grin1 fl/fl mice was 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/+ ; Grin1 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

    Techniques Used: Mouse Assay, Expressing, Western Blot

    The cell membrane expression of EPHB2 was absent in Emx1 cre/+ ; Grin1 fl/fl ; Rosa26 fs-tdTomato positive cells. ( A ) We crossed Emx1 cre/+ ; Grin1 fl/fl mice with Cre-reporter Rosa26 fs-tdTomato mice to produce GluN1 knockout cells labeled with red fluorescence. ( B ) 12 µm coronal brain sections from P8 Emx1 cre/+ ; Grin1 wt/wt ; Rosa26 fs-tdTomato and Emx1 cre/+ ; Grin1 fl/fl ; Rosa26 fs-tdTomato mice of the same litter. Immunostaining of EPHB2 was done in all brain sections. Broad arrowheads pointed the cells with Cre recombination. Triangle arrowheads pointed to cells with Cre recombination. V shaped arrowheads pointed to cells without Cre recombination. In control Emx1 cre/+ ; Grin1 wt/wt ; Rosa26 fs-tdTomato mice, EPHB2 signals were detected on both Cre recombination and non-Cre recombination cells. However, in Emx1 cre/+ ; Grin1 fl/fl ; Rosa26 fs-tdTomato mice, EPHB2 signals were only detected on non-Cre recombination cells, but absent on Cre recombination cells with deletion of GluN1. ( C ) Quantification of fluorescence density of EPHB2 immunostaining for each cell. Compared to control ( Emx1 cre/+ ; Grin1 wt/wt ; Rosa26 fs-tdTomato mice), the protein expression of EPHB2 in Cre recombination cells in Emx1 cre/+ ; Grin1 fl/fl ; Rosa26 fs-tdTomato mice was dramatically decreased. p
    Figure Legend Snippet: The cell membrane expression of EPHB2 was absent in Emx1 cre/+ ; Grin1 fl/fl ; Rosa26 fs-tdTomato positive cells. ( A ) We crossed Emx1 cre/+ ; Grin1 fl/fl mice with Cre-reporter Rosa26 fs-tdTomato mice to produce GluN1 knockout cells labeled with red fluorescence. ( B ) 12 µm coronal brain sections from P8 Emx1 cre/+ ; Grin1 wt/wt ; Rosa26 fs-tdTomato and Emx1 cre/+ ; Grin1 fl/fl ; Rosa26 fs-tdTomato mice of the same litter. Immunostaining of EPHB2 was done in all brain sections. Broad arrowheads pointed the cells with Cre recombination. Triangle arrowheads pointed to cells with Cre recombination. V shaped arrowheads pointed to cells without Cre recombination. In control Emx1 cre/+ ; Grin1 wt/wt ; Rosa26 fs-tdTomato mice, EPHB2 signals were detected on both Cre recombination and non-Cre recombination cells. However, in Emx1 cre/+ ; Grin1 fl/fl ; Rosa26 fs-tdTomato mice, EPHB2 signals were only detected on non-Cre recombination cells, but absent on Cre recombination cells with deletion of GluN1. ( C ) Quantification of fluorescence density of EPHB2 immunostaining for each cell. Compared to control ( Emx1 cre/+ ; Grin1 wt/wt ; Rosa26 fs-tdTomato mice), the protein expression of EPHB2 in Cre recombination cells in Emx1 cre/+ ; Grin1 fl/fl ; Rosa26 fs-tdTomato mice was dramatically decreased. p

    Techniques Used: Expressing, Mouse Assay, Knock-Out, Labeling, Fluorescence, Immunostaining

    The efficiency of intraventricular antibody injection and the distribution territory in the cortex after 3 hr of last injection. ( A ) Anti-GluN1 antibodies were injected into the lateral ventricle from P2 to P8 and mice were perfused 3 hr later after last injection. Rabbit IgG served as control. Mouse brains then were stained with anti-Rabbit secondary coupled with Alexa594. The red fluorophore of Alexa594 indicated where the antibodies had distributed to. Scale bar: 500 μm 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-GluN1 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 B , C ; 200 μm for B’ , C’ . CC: corpus callosum; cg: cingulum; Hip: hippocampus; M: motor cortex; S1: primary somatosensory cortex; S2: secondary somatosensory cortex.
    Figure Legend Snippet: The efficiency of intraventricular antibody injection and the distribution territory in the cortex after 3 hr of last injection. ( A ) Anti-GluN1 antibodies were injected into the lateral ventricle from P2 to P8 and mice were perfused 3 hr later after last injection. Rabbit IgG served as control. Mouse brains then were stained with anti-Rabbit secondary coupled with Alexa594. The red fluorophore of Alexa594 indicated where the antibodies had distributed to. Scale bar: 500 μm 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-GluN1 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 B , C ; 200 μm for B’ , C’ . CC: corpus callosum; cg: cingulum; Hip: hippocampus; M: motor cortex; S1: primary somatosensory cortex; S2: secondary somatosensory cortex.

    Techniques Used: Injection, Mouse Assay, Staining, Fluorescence

    2) Product Images from "NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling"

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

    Journal: bioRxiv

    doi: 10.1101/2020.06.03.130559

    The efficiency of intraventricular antibody injection and the distribution territory in the cortex after 3hr of last injection. (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.
    Figure Legend Snippet: The efficiency of intraventricular antibody injection and the distribution territory in the cortex after 3hr of last injection. (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.

    Techniques Used: Injection, Mouse Assay, Staining, Fluorescence

    NMDAR is required in target neurons for normal callosal innervation. (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.
    Figure Legend Snippet: NMDAR is required in target neurons for normal callosal innervation. (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.

    Techniques Used: Expressing, Mouse Assay, In Utero, Electroporation, Fluorescence, Labeling

    Emx1 cre/+ ; NR2B fl/fl but not Emx1 cre/+ ; NR2A fl/fl mice had the same disrupted callosal innervation patterns as Emx1 cre/+ ; NR1 fl/fl at P14. Callosal innervation patterns in control Emx1 cre/+ ; NR2A fl/wt (A) and Emx1 cre/+ ; NR2A fl/fl mice (B) at P14. “*” points out M1/S1 border. (C) Quantification of fluorescence density. P = 0.392. Callosal innervation patterns in control Emx1 cre/+ ; NR2B fl/wt (D) and Emx1 cre/+ ; NR2B fl/fl mice (E) at P14. (F) Quantification of fluorescence density. P = 0.03. Scale bar: 500μm for all images.
    Figure Legend Snippet: Emx1 cre/+ ; NR2B fl/fl but not Emx1 cre/+ ; NR2A fl/fl mice had the same disrupted callosal innervation patterns as Emx1 cre/+ ; NR1 fl/fl at P14. Callosal innervation patterns in control Emx1 cre/+ ; NR2A fl/wt (A) and Emx1 cre/+ ; NR2A fl/fl mice (B) at P14. “*” points out M1/S1 border. (C) Quantification of fluorescence density. P = 0.392. Callosal innervation patterns in control Emx1 cre/+ ; NR2B fl/wt (D) and Emx1 cre/+ ; NR2B fl/fl mice (E) at P14. (F) Quantification of fluorescence density. P = 0.03. Scale bar: 500μm for all images.

    Techniques Used: Mouse Assay, Fluorescence

    NR2B ( Emx1 cre/+ ; NR2B fl/fl ) but not NR2A ( Emx1 cre/+ ; NR2A fl/fl ) mice had same disrupted callosal innervation patterns as Emx1 cre/+ ; NR1 fl/fl at P30. (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.
    Figure Legend Snippet: NR2B ( Emx1 cre/+ ; NR2B fl/fl ) but not NR2A ( Emx1 cre/+ ; NR2A fl/fl ) mice had same disrupted callosal innervation patterns as Emx1 cre/+ ; NR1 fl/fl at P30. (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.

    Techniques Used: Mouse Assay, Mutagenesis, Fluorescence

    The expression of NMDAR in cortex was reduced in Emx1 cre/+ ; NR1 fl/fl mice. 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.
    Figure Legend Snippet: The expression of NMDAR in cortex was reduced in Emx1 cre/+ ; NR1 fl/fl mice. 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.

    Techniques Used: Expressing, Mouse Assay, Immunostaining, Staining

    The callosal somatosensory innervation pattern was disrupted in Emx1 cre/+ ; NR1 fl/fl mice. (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.
    Figure Legend Snippet: The callosal somatosensory innervation pattern was disrupted in Emx1 cre/+ ; NR1 fl/fl mice. (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.

    Techniques Used: Mouse Assay, Plasmid Preparation, Injection, Fluorescence

    No increased cell death in S1 of Emx1 cre/+ ; NR1 fl/fl mice at P6. (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’’.
    Figure Legend Snippet: No increased cell death in S1 of Emx1 cre/+ ; NR1 fl/fl mice at P6. (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’’.

    Techniques Used: Mouse Assay, Mutagenesis

    Increased callosal innervation in S1 after contralateral but not ipsilateral injection of anti-NMDAR antibodies from P2 to P12. (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.
    Figure Legend Snippet: Increased callosal innervation in S1 after contralateral but not ipsilateral injection of anti-NMDAR antibodies from P2 to P12. (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.

    Techniques Used: Injection, Mouse Assay, Fluorescence

    Deleting NMDAR in upper contralateral cortical layers had no effect on callosal innervation pattern of S1 at P14. (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.
    Figure Legend Snippet: Deleting NMDAR in upper contralateral cortical layers had no effect on callosal innervation pattern of S1 at P14. (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.

    Techniques Used: In Utero, Electroporation, Mouse Assay, Labeling, Fluorescence

    There was no difference between Emx1 cre/+ ; NR1 fl/wt and Emx1 cre/+ ; NR1 fl/fl mice during axonal extension into the ipsilateral CC (P0) and to the contralateral CC (P5). (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.
    Figure Legend Snippet: There was no difference between Emx1 cre/+ ; NR1 fl/wt and Emx1 cre/+ ; NR1 fl/fl mice during axonal extension into the ipsilateral CC (P0) and to the contralateral CC (P5). (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.

    Techniques Used: Mouse Assay

    The protein but not RNA level of EPHB2 was reduced in Emx1 cre/+ ; NR1 fl/fl mice at P8. (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
    Figure Legend Snippet: The protein but not RNA level of EPHB2 was reduced in Emx1 cre/+ ; NR1 fl/fl mice at P8. (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

    Techniques Used: Mouse Assay, Expressing, Western Blot

    Contralateral injection of anti-NMDAR antibodies from P4 to P8 but not P8 to P14 had increased callosal innervation in S1. (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.
    Figure Legend Snippet: Contralateral injection of anti-NMDAR antibodies from P4 to P8 but not P8 to P14 had increased callosal innervation in S1. (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.

    Techniques Used: Injection, Mouse Assay, Fluorescence

    The callosal innervation defect was first detected at P6 in Emx1 cre/+ ; NR1 fl/fl mice. (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.
    Figure Legend Snippet: The callosal innervation defect was first detected at P6 in Emx1 cre/+ ; NR1 fl/fl mice. (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.

    Techniques Used: Mouse Assay, Mutagenesis, Fluorescence

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    Alomone Labs rabbit anti nr1
    Exposure to high glucose (HG) increases expression of NMDA receptor subunits in primary cultures of rat mesangial cells. A : Representative results of RT-PCR showing significantly increased abundance of transcripts encoding <t>NR1,</t> NR2B, and NR2C subunits but not of NR2A or NR2D in cells cultured for 24 h in HG medium compared with cells cultured in normal glucose (control [Con]). B : Densitometric analysis of three repetitions of the experiments shown in A . C : Immunoblot analysis showing increased abundance of NMDA receptor subunits in primary cultures of rat mesangial cells cultured in HG. D : Densitometric analysis of three repetitions of the experiments shown in C . Data are mean ± SD. * P
    Rabbit Anti Nr1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti nr1/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
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    93
    Alomone Labs immunostaining rabbit anti nr1
    The expression of NMDAR in cortex was reduced in Emx1 cre/+ ; <t>NR1</t> fl/fl mice. 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. <t>Immunostaining</t> 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.
    Immunostaining Rabbit Anti Nr1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/immunostaining rabbit anti nr1/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Image Search Results


    Exposure to high glucose (HG) increases expression of NMDA receptor subunits in primary cultures of rat mesangial cells. A : Representative results of RT-PCR showing significantly increased abundance of transcripts encoding NR1, NR2B, and NR2C subunits but not of NR2A or NR2D in cells cultured for 24 h in HG medium compared with cells cultured in normal glucose (control [Con]). B : Densitometric analysis of three repetitions of the experiments shown in A . C : Immunoblot analysis showing increased abundance of NMDA receptor subunits in primary cultures of rat mesangial cells cultured in HG. D : Densitometric analysis of three repetitions of the experiments shown in C . Data are mean ± SD. * P

    Journal: Diabetes

    Article Title: NMDA Receptors as Potential Therapeutic Targets in Diabetic Nephropathy: Increased Renal NMDA Receptor Subunit Expression in Akita Mice and Reduced Nephropathy Following Sustained Treatment With Memantine or MK-801

    doi: 10.2337/db16-0209

    Figure Lengend Snippet: Exposure to high glucose (HG) increases expression of NMDA receptor subunits in primary cultures of rat mesangial cells. A : Representative results of RT-PCR showing significantly increased abundance of transcripts encoding NR1, NR2B, and NR2C subunits but not of NR2A or NR2D in cells cultured for 24 h in HG medium compared with cells cultured in normal glucose (control [Con]). B : Densitometric analysis of three repetitions of the experiments shown in A . C : Immunoblot analysis showing increased abundance of NMDA receptor subunits in primary cultures of rat mesangial cells cultured in HG. D : Densitometric analysis of three repetitions of the experiments shown in C . Data are mean ± SD. * P

    Article Snippet: Primary antibodies were rabbit anti-NR1 (AGC-001 1:100; Alomone Labs) or rabbit anti-NR2A (AGC-002 1:100; Alomone Labs) for 24 h at 4°C.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture

    Increased expression of NMDA receptor subunits in renal cortex of Akita mice. A : Representative results of RT-PCR showing increased abundance of transcripts encoding NR1, NR2A, and NR2C subunits but not in NR2B or NR2D in renal cortex in 12-week-old Akita mice compared with 12-week-old DBA/2J control mice. B : Densitometric analysis from four mice per group. C : Immunoblot analysis showing increased abundance of NMDA receptor subunits in Akita mice compared with DBA/2J control mice. D : Densitometric analysis from four mice per group. Data are mean ± SD. * P

    Journal: Diabetes

    Article Title: NMDA Receptors as Potential Therapeutic Targets in Diabetic Nephropathy: Increased Renal NMDA Receptor Subunit Expression in Akita Mice and Reduced Nephropathy Following Sustained Treatment With Memantine or MK-801

    doi: 10.2337/db16-0209

    Figure Lengend Snippet: Increased expression of NMDA receptor subunits in renal cortex of Akita mice. A : Representative results of RT-PCR showing increased abundance of transcripts encoding NR1, NR2A, and NR2C subunits but not in NR2B or NR2D in renal cortex in 12-week-old Akita mice compared with 12-week-old DBA/2J control mice. B : Densitometric analysis from four mice per group. C : Immunoblot analysis showing increased abundance of NMDA receptor subunits in Akita mice compared with DBA/2J control mice. D : Densitometric analysis from four mice per group. Data are mean ± SD. * P

    Article Snippet: Primary antibodies were rabbit anti-NR1 (AGC-001 1:100; Alomone Labs) or rabbit anti-NR2A (AGC-002 1:100; Alomone Labs) for 24 h at 4°C.

    Techniques: Expressing, Mouse Assay, Reverse Transcription Polymerase Chain Reaction

    Immunohistochemistry (IHC) suggests increased abundance of NMDA receptor subunits throughout the kidney of 12-week-old Akita mice. IHC was carried out in paraffin sections. Negative control sections shown at the top were not exposed to a primary antibody. A : Especially large increases in NR1, NR2A, and NR2C in renal tubules. B : Signal in glomeruli for NR1, NR2A, and NR2C. Primary processes were visible in some of the cells within glomeruli. C : Staining intensity per square micron in the whole kidney (top) and within glomeruli (bottom). Data are mean ± SD. * P

    Journal: Diabetes

    Article Title: NMDA Receptors as Potential Therapeutic Targets in Diabetic Nephropathy: Increased Renal NMDA Receptor Subunit Expression in Akita Mice and Reduced Nephropathy Following Sustained Treatment With Memantine or MK-801

    doi: 10.2337/db16-0209

    Figure Lengend Snippet: Immunohistochemistry (IHC) suggests increased abundance of NMDA receptor subunits throughout the kidney of 12-week-old Akita mice. IHC was carried out in paraffin sections. Negative control sections shown at the top were not exposed to a primary antibody. A : Especially large increases in NR1, NR2A, and NR2C in renal tubules. B : Signal in glomeruli for NR1, NR2A, and NR2C. Primary processes were visible in some of the cells within glomeruli. C : Staining intensity per square micron in the whole kidney (top) and within glomeruli (bottom). Data are mean ± SD. * P

    Article Snippet: Primary antibodies were rabbit anti-NR1 (AGC-001 1:100; Alomone Labs) or rabbit anti-NR2A (AGC-002 1:100; Alomone Labs) for 24 h at 4°C.

    Techniques: Immunohistochemistry, Mouse Assay, Negative Control, Staining

    Exposure to high glucose (HG) increases expression of NMDA receptor subunits in cultured mouse podocytes (MPC-5 cells). A : Representative results of RT-PCR showing increased abundance of transcripts encoding NR1, NR2A, NR2B, and NR2C subunits in cells cultured for 24 h in a medium containing 25 mmol/L glucose (HG). There was no change in NR2D. Control cells (Con) were cultured in medium containing 9 mmol/L glucose, with 16 mmol/L mannitol as an osmotic control. B : Densitometric analysis of three repetitions of the experiments shown in A . C : Immunoblot analysis showing increased abundance of NMDA receptor subunits in podocytes cultured in HG compared with Con. D : Densitometric analysis of three repetitions of the experiments shown in C . Data are mean ± SD. * P

    Journal: Diabetes

    Article Title: NMDA Receptors as Potential Therapeutic Targets in Diabetic Nephropathy: Increased Renal NMDA Receptor Subunit Expression in Akita Mice and Reduced Nephropathy Following Sustained Treatment With Memantine or MK-801

    doi: 10.2337/db16-0209

    Figure Lengend Snippet: Exposure to high glucose (HG) increases expression of NMDA receptor subunits in cultured mouse podocytes (MPC-5 cells). A : Representative results of RT-PCR showing increased abundance of transcripts encoding NR1, NR2A, NR2B, and NR2C subunits in cells cultured for 24 h in a medium containing 25 mmol/L glucose (HG). There was no change in NR2D. Control cells (Con) were cultured in medium containing 9 mmol/L glucose, with 16 mmol/L mannitol as an osmotic control. B : Densitometric analysis of three repetitions of the experiments shown in A . C : Immunoblot analysis showing increased abundance of NMDA receptor subunits in podocytes cultured in HG compared with Con. D : Densitometric analysis of three repetitions of the experiments shown in C . Data are mean ± SD. * P

    Article Snippet: Primary antibodies were rabbit anti-NR1 (AGC-001 1:100; Alomone Labs) or rabbit anti-NR2A (AGC-002 1:100; Alomone Labs) for 24 h at 4°C.

    Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction

    The expression of NMDAR in cortex was reduced in Emx1 cre/+ ; NR1 fl/fl mice. 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.

    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: The expression of NMDAR in cortex was reduced in Emx1 cre/+ ; NR1 fl/fl mice. 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 immunostaining : Rabbit anti-NR1 (1:500, AGC-001, Alomone labs), anti-vGlut2 (1:200, AB2251, Millipore), goat anti-EphB2 (1:50, AF467, R & D), anti-cleaved caspase-3 ( # 9661S, Cell Signaling), anti-Rabbit 594 (#711-585-152, Jackson ImmunoResearch), and anti-guinea pig 488 (A-11073, Invitrogen).

    Techniques: Expressing, Mouse Assay, Immunostaining, Staining

    SHP2 D61G overexpression at DIV 12 increases surface AMPA receptor expression. (A-B) Representative images of surface GluN1 (A) or GluA1 (B) receptor immunostaining. GFP alone (upper) or SHP2 D61G and GFP (lower) were expressed in cultured hippocampal neuron at DIV 12. Scale bar, 20 μm. (C) Bar graph showing the average size (left) or the number (right) of GluN1-stained particles per 10 μm of dendrites. Unpaired t -test, ** P

    Journal: Neuroscience letters

    Article Title: Noonan syndrome-associated SHP2 mutation differentially modulates the expression of postsynaptic receptors according to developmental maturation

    doi: 10.1016/j.neulet.2017.03.036

    Figure Lengend Snippet: SHP2 D61G overexpression at DIV 12 increases surface AMPA receptor expression. (A-B) Representative images of surface GluN1 (A) or GluA1 (B) receptor immunostaining. GFP alone (upper) or SHP2 D61G and GFP (lower) were expressed in cultured hippocampal neuron at DIV 12. Scale bar, 20 μm. (C) Bar graph showing the average size (left) or the number (right) of GluN1-stained particles per 10 μm of dendrites. Unpaired t -test, ** P

    Article Snippet: The following antibodies were used: polyclonal anti-GluN1 (extracellular) antibody (#AGC-001, alomone labs, Jerusalem, Israel), polyclonal anti-GluA1 (extracellular) antibody (#AGC-004, alomone labs), Cy™3-conjugated goat anti-rabbit IgG antibody (#111–165-003, Jackson ImmunoResearch Lab, West Grove, PA).0Images were acquired by using confocal microscope (Zeiss 710, Carl Zeiss, Oberkochen, Germany) and analyzed by using NIH ImageJ software (ver.

    Techniques: Over Expression, Expressing, Immunostaining, Cell Culture, Staining

    SHP2 D61G overexpression at DIV 6 increases surface NMDA receptor expression. (A-B) Representative images of surface GluN1 (A) or GluA1 (B) receptor immunostaining. GFP alone (upper) or SHP2 D61G and GFP (lower) were expressed in cultured hippocampal neuron at DIV6 by using a bicistronic Sindbis virus vector. Scale bar, 20 μm. (C) Bar graph showing the average size (left) or the number (right) of GluN1-stained particles per 10 μm of dendrites. Unpaired t -test, ns, not significant. (D) Bar graph showing the average size (left) or the number (right) of GluA1-stained particles per 10 μm of dendrites. Unpaired t -test, * P

    Journal: Neuroscience letters

    Article Title: Noonan syndrome-associated SHP2 mutation differentially modulates the expression of postsynaptic receptors according to developmental maturation

    doi: 10.1016/j.neulet.2017.03.036

    Figure Lengend Snippet: SHP2 D61G overexpression at DIV 6 increases surface NMDA receptor expression. (A-B) Representative images of surface GluN1 (A) or GluA1 (B) receptor immunostaining. GFP alone (upper) or SHP2 D61G and GFP (lower) were expressed in cultured hippocampal neuron at DIV6 by using a bicistronic Sindbis virus vector. Scale bar, 20 μm. (C) Bar graph showing the average size (left) or the number (right) of GluN1-stained particles per 10 μm of dendrites. Unpaired t -test, ns, not significant. (D) Bar graph showing the average size (left) or the number (right) of GluA1-stained particles per 10 μm of dendrites. Unpaired t -test, * P

    Article Snippet: The following antibodies were used: polyclonal anti-GluN1 (extracellular) antibody (#AGC-001, alomone labs, Jerusalem, Israel), polyclonal anti-GluA1 (extracellular) antibody (#AGC-004, alomone labs), Cy™3-conjugated goat anti-rabbit IgG antibody (#111–165-003, Jackson ImmunoResearch Lab, West Grove, PA).0Images were acquired by using confocal microscope (Zeiss 710, Carl Zeiss, Oberkochen, Germany) and analyzed by using NIH ImageJ software (ver.

    Techniques: Over Expression, Expressing, Immunostaining, Cell Culture, Plasmid Preparation, Staining

    CIE does not impact the expression levels of proteins commonly associated with alterations in excitatory neurotransmission in the vHC. However, in the dHC, the GluA1 subunit is upregulated. Representative western blot examples illustrating the expression of (A) the GluA1 and (B) GluA2 subunit of the AMPA receptor, (E) the GluN1, (F) and GluN2 subunit of the NMDA receptor and (G) SK2. In panels (A,B,E–G) the corresponding expression of actin (normalization protein) is shown in the lower blot panel of each protein of interest. (C,D,H–J) Group data, normalized to actin and the internal control group (Air), for the (C) GluA1 (Air n = 4, CIE n = 4), (D) GluA2 (Air n = 4, CIE n = 4), (H) GluN1 (Air n = 4, CIE n = 4), (I) GluN2B subunit (Air n = 4, CIE n = 4) and (J) the SK2 channel (Air n = 4, CIE n = 4) illustrating an increase in the expression of the GluA1 subunit (Air n = 4, CIE n = 4) in the dHC without changes in other proteins in the dHC and vHC of Air and CIE-treated rats. All data are expressed as the mean ± SEM, * p

    Journal: Frontiers in Neuroscience

    Article Title: Chronic Ethanol Exposures Leads to a Negative Affective State in Female Rats That Is Accompanied by a Paradoxical Decrease in Ventral Hippocampus Excitability

    doi: 10.3389/fnins.2021.669075

    Figure Lengend Snippet: CIE does not impact the expression levels of proteins commonly associated with alterations in excitatory neurotransmission in the vHC. However, in the dHC, the GluA1 subunit is upregulated. Representative western blot examples illustrating the expression of (A) the GluA1 and (B) GluA2 subunit of the AMPA receptor, (E) the GluN1, (F) and GluN2 subunit of the NMDA receptor and (G) SK2. In panels (A,B,E–G) the corresponding expression of actin (normalization protein) is shown in the lower blot panel of each protein of interest. (C,D,H–J) Group data, normalized to actin and the internal control group (Air), for the (C) GluA1 (Air n = 4, CIE n = 4), (D) GluA2 (Air n = 4, CIE n = 4), (H) GluN1 (Air n = 4, CIE n = 4), (I) GluN2B subunit (Air n = 4, CIE n = 4) and (J) the SK2 channel (Air n = 4, CIE n = 4) illustrating an increase in the expression of the GluA1 subunit (Air n = 4, CIE n = 4) in the dHC without changes in other proteins in the dHC and vHC of Air and CIE-treated rats. All data are expressed as the mean ± SEM, * p

    Article Snippet: The following antibodies were used to visualize the proteins of interest: mouse anti-GluA1 (1:1,000; Neuromab; 75-327); mouse anti-GluA2 (1:1,000; Neuromab; 75-002); rabbit anti-SK2 (1:1,000; Alomone Lab, APC-028); rabbit anti-GluN1 (1:1,000; Alomone Labs; AGC-001); rabbit anti-GluN2B (1:500; Alomone Labs, AGC-003); rabbit anti-GABAAR α1 subunit (1:1,000; Novus, NB300-191) mouse anti-Gephyrin (1:1,000, Synaptic Systems, 147-011).

    Techniques: Expressing, Western Blot