Journal: Molecular Brain

Article Title: The production of viral vectors designed to express large and difficult to express transgenes within neurons

doi: 10.1186/s13041-015-0100-7

Figure Lengend Snippet: Adeno-associated viral vector maps and GluN2 and GFP transgene expression from these AAV viral vector plasmids in vitro . (A) Viral genome maps for pAAV-Basic vector backbones containing the following transgenes: Flag-GluN2A/B, Flag-GluN2A/B with C-terminal deletions (GluN2A/BΔC) and GFP. These transgenes are controlled by the 0.4αCaMKII promoter or CMV promoter as indicated. Each of these vectors contain a ~200 bps 3′UTR that contains an SV40 based poly-adenylation signal. GFP = green fluorescence protein, ITR = inverted terminal repeat, MCS = multiple cloning site (B) AAV plasmids designed to express, Flag-GluN2A/B, GFP, and Flag-GluN2A/BΔC from a 0.4αCaMKII promoter and Flag-GluN2A/BΔC from a CMV promoter were transfected into N2A cells. Twenty four hours after transfection, native GFP expression was observed via fluorescence microscopy and Flag-GluN2 expression was observed via ICC and fluorescence microscopy. Images depict DAPI stained nuclei with the same fields viewed for GFP or Flag-GluN2 (Texas Red) transgene expression. Flag-GluN2A/B did not exhibit detectable expression. However GFP and Flag-GluN2A/BΔC exhibited convincing expression. Untransfected cells and cells transfected with pRK5-Flag-GluN2 plasmids were processed as negative and positive ICC controls respectively. (Scale bar = 20 μm).

Article Snippet: Purified viruses were titered using a qRT-PCR based method to determine the number of DNAse resistant viral particles and the results were reported as genomes copies (GC)/mL and this was performed as previously described [ ] using PCR primers specific for GluN2A ( Life Technologies , CatRn00561341_m1), GluN2B ( Life Technologies , Cat# Mm00433820_m1) and GFP ( Life technologies , Cat# Mr04329676_mr).

Techniques: Plasmid Preparation, Expressing, In Vitro, Fluorescence, Cloning, Transfection, Microscopy, Staining

Journal: Molecular Brain

Article Title: The production of viral vectors designed to express large and difficult to express transgenes within neurons

doi: 10.1186/s13041-015-0100-7

Figure Lengend Snippet: Vector maps of AAV vectors optimized for large transgenes. Viral genome maps for pAAV vector backbones containing either the Flag-GluN2A/B or GFP transgenes. Each of these vectors contain a ~75 bps 3′UTR that contains an SV40 based poly-adenylation signal. These transgenes are controlled by one of the following promoters as indicated: 1.3αCaMKII, 0.4αCaMKII, 1.1Synapsin, 0.5Synapsin, CMV, TRE3G, EFS, or RSV. The promoters are depicted as their approximate relative sizes to one another. GFP = green fluorescence protein, ITR = inverted terminal repeat, MCS = multiple cloning site.

Article Snippet: Purified viruses were titered using a qRT-PCR based method to determine the number of DNAse resistant viral particles and the results were reported as genomes copies (GC)/mL and this was performed as previously described [ ] using PCR primers specific for GluN2A ( Life Technologies , CatRn00561341_m1), GluN2B ( Life Technologies , Cat# Mm00433820_m1) and GFP ( Life technologies , Cat# Mr04329676_mr).

Techniques: Plasmid Preparation, Fluorescence, Cloning

Journal: Molecular Brain

Article Title: The production of viral vectors designed to express large and difficult to express transgenes within neurons

doi: 10.1186/s13041-015-0100-7

Figure Lengend Snippet: GluN2 and GFP transgene expression in vitro from optimized AAV vector plasmids. Representative ICC images from transfections with optimized AAV plasmids designed to express Flag-GluN2A/B or GFP transgenes utilizing one of the following promoters: 1.3αCaMKII, 0.4αCaMKII, 1.1Synapsin, 0.5Synapsin, CMV, TRE3G, EFS, or RSV. Plasmids containing transgenes controlled by the neuron specific, 0.4αCaMKII, 1.3αCaMKII, 0.5synapsin and 1.1synapsin promoters were transfected into N2A cells. Plasmids containing transgenes controlled by CMV, TRE3G, EFS and RSV promoters were transfected into 293FT cells. TRE3G promoter containing plasmids were co-transfected with the pTet-Off plasmid. Twenty four hours after transfection, native GFP expression was observed via fluorescence microscopy and Flag-GluN2 expression was observed via ICC and fluorescence microscopy. Images depict DAPI stained nuclei with the same fields viewed for GFP or Flag-GluN2 (Texas Red) transgene expression. Plasmids with neuron specific promoters were able to confer GFP expression but were not able to confer GluN2 expression. Plasmids containing transgenes controlled by CMV, TRE3G, EFS and RSV promoters were capable of conferring GluN2 and GFP expression. Untransfected cells and cells transfected with pRK5-Flag-GluN2 plasmids were processed as negative and positive ICC controls respectively. (Scale bar = 20 μm).

Article Snippet: Purified viruses were titered using a qRT-PCR based method to determine the number of DNAse resistant viral particles and the results were reported as genomes copies (GC)/mL and this was performed as previously described [ ] using PCR primers specific for GluN2A ( Life Technologies , CatRn00561341_m1), GluN2B ( Life Technologies , Cat# Mm00433820_m1) and GFP ( Life technologies , Cat# Mr04329676_mr).

Techniques: Expressing, In Vitro, Plasmid Preparation, Transfection, Fluorescence, Microscopy, Staining

Journal: Molecular Brain

Article Title: The production of viral vectors designed to express large and difficult to express transgenes within neurons

doi: 10.1186/s13041-015-0100-7

Figure Lengend Snippet: The CMV, TRE3G, EFS and RSV promoters differ in their ability to confer GluN2A expression in vitro . (A) Representative ICC images for Flag-GluN2A expression and associated GFP and DAPI staining from transfected cells. In this experiment, AAV plasmids designed to express GluN2A from one of the following promoters, (CMV, TRE3G, EFS, RSV), were cotransfected into 293FT cells with a plasmid containing a CMV-GFP transgene and the pTet-Off plasmid. Twenty four hours after transfection, native GFP expression was observed via fluorescence microscopy and Flag-GluN2 expression was observed via ICC and fluorescence microscopy. Images depict DAPI stained nuclei with the same fields viewed for GFP and Flag-GluN2 (Texas Red) transgene expression. GFP was used as a transfection efficiency control. (B) Quantitation of Flag-GluN2A expression data presented in (A) . The Texas Red (Flag-GluN2A) expression levels were normalized to GFP expression levels and these data were plotted as average percent expression as compared to the control group. n = 6, Error bars represent standard error of the mean (SEM). The AAV-CMV-GluN2A plasmid conferred similar Flag-GluN2A expression as the positive control (pRK5-Flag-GluN2A) and these exhibited higher levels of Flag-GluN2A expression, as compared to TRE3G, EFS, and RSV GluN2A containing plasmids. (C) p values for one-way ANOVA with Fisher’s PLSD post-hoc test for (B) . Differences were considered significant if, p < 0.05. (D) Mean OD for GFP expression and DAPI staining for Flag-GluN2A expression data presented in (A) . One-way ANOVA revealed that GFP levels (F(4,25) = 1.532; p = 0.2235) and DAPI levels (F(4,25 = 1.652; p = 0.1925), did not differ significantly indicating that transfection efficiency did not differ among the groups and the number of cells quantified did not differ significantly among the groups.

Article Snippet: Purified viruses were titered using a qRT-PCR based method to determine the number of DNAse resistant viral particles and the results were reported as genomes copies (GC)/mL and this was performed as previously described [ ] using PCR primers specific for GluN2A ( Life Technologies , CatRn00561341_m1), GluN2B ( Life Technologies , Cat# Mm00433820_m1) and GFP ( Life technologies , Cat# Mr04329676_mr).

Techniques: Expressing, In Vitro, Staining, Transfection, Plasmid Preparation, Fluorescence, Microscopy, Control, Quantitation Assay, Positive Control, IF-P

Journal: Molecular Brain

Article Title: The production of viral vectors designed to express large and difficult to express transgenes within neurons

doi: 10.1186/s13041-015-0100-7

Figure Lengend Snippet: GluN2 and GFP transgene expression in vitro mediated by adeno-associated virus. (A) In this experiment, 293FT cells were transduced with AAV viruses designed to express GFP from a CMV promoter, Flag-GluN2A from the CMV, TRE3G, EFS, or RSV promoters, and Flag-GluN2B and GluN2A/BΔC from a CMV promoter. TRE3G promoter containing viruses were transduced into cells that were also transfected with the pTet-Off plasmid. Forty eight hours after viral transduction, native GFP expression was observed via fluorescence microscopy and Flag-GluN2 expression was observed via ICC and fluorescence microscopy. Images depict DAPI stained nuclei with the same fields viewed for GFP or Flag-GluN2 (Texas Red) transgene expression. Non-transduced cells and cells transfected with pRK5-Flag-GluN2 plasmids were processed as negative and positive ICC controls respectively. AAV designed to express Flag-GluN2 from the CMV, TRE3G, EFS, or RSV promoters did not confer convincing transgene expression. However AAV designed to express GFP or GluN2A/BΔC exhibited expression in > 90% of cells. (Scale bar = 20 μm) (B) Depicts similar images as depicted in A, but at lower magnification. (Scale bar = 50 μm).

Article Snippet: Purified viruses were titered using a qRT-PCR based method to determine the number of DNAse resistant viral particles and the results were reported as genomes copies (GC)/mL and this was performed as previously described [ ] using PCR primers specific for GluN2A ( Life Technologies , CatRn00561341_m1), GluN2B ( Life Technologies , Cat# Mm00433820_m1) and GFP ( Life technologies , Cat# Mr04329676_mr).

Techniques: Expressing, In Vitro, Virus, Transduction, Transfection, Plasmid Preparation, Fluorescence, Microscopy, Staining

Journal: Molecular Brain

Article Title: The production of viral vectors designed to express large and difficult to express transgenes within neurons

doi: 10.1186/s13041-015-0100-7

Figure Lengend Snippet: GluN2 and GFP transgene expression in vivo mediated by adeno-associated virus. In this experiment, AAV viruses designed to express Flag-GluN2A/B, GFP, and Flag-GluN2A/BΔC from a CMV, EFS, TRE3G or 0.4αCaMKII promoter, as indicated, were infused into the basolateral complex of the amygdala (BLA). All viruses were infused into the rat BLA, except for the TRE3G promoter containing viruses – these viruses were infused into the BLA of αCaMKII-tTA transgenic mice. Twenty one days following viral infusion, coronal sections were prepared that contained the BLA and native GFP expression was observed via fluorescence microscopy and Flag-GluN2 expression was observed via immunohistochemistry(IHC) and fluorescence microscopy. Images depict DAPI stained nuclei with the same fields viewed for GFP or Flag-GluN2 (Texas Red) transgene expression. Viruses designed to express GFP and Flag-GluN2A/BΔC exhibited convincing and robust transgene expression in vivo . Viruses designed to express Flag-GluN2A/B were not capable of conferring GluN2 expression in vivo . *indicates virus derived from pAAV-Basic vector. Coronal sections from naïve controls were processed as negative controls for Flag-IHC and coronal sections from wild type mice were processed as controls (Control A) for mice infused with AAV-TRE3G-Flag-GluN2A.

Article Snippet: Purified viruses were titered using a qRT-PCR based method to determine the number of DNAse resistant viral particles and the results were reported as genomes copies (GC)/mL and this was performed as previously described [ ] using PCR primers specific for GluN2A ( Life Technologies , CatRn00561341_m1), GluN2B ( Life Technologies , Cat# Mm00433820_m1) and GFP ( Life technologies , Cat# Mr04329676_mr).

Techniques: Expressing, In Vivo, Virus, Transgenic Assay, Fluorescence, Microscopy, Immunohistochemistry, Staining, Derivative Assay, Plasmid Preparation, Control

Journal: Molecular Brain

Article Title: The production of viral vectors designed to express large and difficult to express transgenes within neurons

doi: 10.1186/s13041-015-0100-7

Figure Lengend Snippet: Lentiviral vector maps. Viral genome maps for pLenti7.3 vector backbones containing either the Flag-GluN2A/B or GFP transgenes. These transgenes are controlled by one of the following promoters as indicated: 1.3αCaMKII, 0.4αCaMKII, 1.1Synapsin, 0.5Synapsin, CMV, or TRE3G. The promoters are depicted as their approximate relative sizes to one another. GFP = green fluorescence protein, LTR = long terminal repeat, MCS = multiple cloning site, WPRE = woodchuck hepatitis post-transcriptional regulatory element, RSV = Rous Sarcoma virus promoter.

Article Snippet: Purified viruses were titered using a qRT-PCR based method to determine the number of DNAse resistant viral particles and the results were reported as genomes copies (GC)/mL and this was performed as previously described [ ] using PCR primers specific for GluN2A ( Life Technologies , CatRn00561341_m1), GluN2B ( Life Technologies , Cat# Mm00433820_m1) and GFP ( Life technologies , Cat# Mr04329676_mr).

Techniques: Plasmid Preparation, Fluorescence, Cloning, Virus

Journal: Molecular Brain

Article Title: The production of viral vectors designed to express large and difficult to express transgenes within neurons

doi: 10.1186/s13041-015-0100-7

Figure Lengend Snippet: GluN2 and GFP transgene expression in vitro from lentiviral vector plasmids. Representative ICC images from transfections with lentiviral plasmids designed to express Flag-GluN2A/B or GFP transgenes utilizing one of the following promoters: 1.3αCaMKII, 0.4αCaMKII, 1.1Synapsin, 0.5Synapsin, CMV, or TRE3G. Plasmids containing transgenes controlled by the neuron specific, 0.4αCaMKII, 1.3αCaMKII, 0.5synapsin and 1.1synapsin promoters were transfected into N2A cells. Plasmids containing transgenes controlled by CMV and TRE3G promoters were transfected into 293FT cells. TRE3G promoter containing plasmids were co-transfected with the pTet-Off plasmid. Twenty four hours after transfection, native GFP expression was observed via fluorescence microscopy and Flag-GluN2 expression was observed via ICC and fluorescence microscopy. Images depict DAPI stained nuclei with the same fields viewed for GFP or Flag-GluN2 (Texas Red) transgene expression. All lentiviral plasmids were capable of conferring GluN2 or GFP expression as intended. Untransfected cells and cells transfected with pRK5-Flag-GluN2 plasmids were processed as negative and positive ICC controls respectively. (Scale bar = 20 μm).

Article Snippet: Purified viruses were titered using a qRT-PCR based method to determine the number of DNAse resistant viral particles and the results were reported as genomes copies (GC)/mL and this was performed as previously described [ ] using PCR primers specific for GluN2A ( Life Technologies , CatRn00561341_m1), GluN2B ( Life Technologies , Cat# Mm00433820_m1) and GFP ( Life technologies , Cat# Mr04329676_mr).

Techniques: Expressing, In Vitro, Plasmid Preparation, Transfection, Fluorescence, Microscopy, Staining

Journal: Molecular Brain

Article Title: The production of viral vectors designed to express large and difficult to express transgenes within neurons

doi: 10.1186/s13041-015-0100-7

Figure Lengend Snippet: Lentiviral plasmids confer spurious transgene expression in vitro , due to the presence of the RSV promoter at the 5′ end of the lentiviral genome. In this experiment, AAV and lentiviral plasmids designed to express Flag-GluN2A/B from a 0.4αCaMKII promoter, and a lentiviral plasmid designed to express Flag-GluN2A from a 0.4αCaMKII promoter which had the RSV promoter deleted (−RSV), were transfected into 293FT cells. Twenty four hours after transfection Flag-GluN2 expression was observed via ICC and fluorescence microscopy. Images depict DAPI stained nuclei with the same fields viewed Flag-GluN2 (Texas Red) transgene expression. Similar to previous results, the AAV plasmids did not confer GluN2 expression and the lentiviral plasmids did confer GluN2 transgene expression. However the lentiviral plasmid without the RSV promoter did not confer 0.4αCaMKII-GluN2A transgene expression. Untransfected cells and cells transfected with pRK5-Flag-GluN2 plasmids were processed as negative and positive ICC controls respectively, (scale bar = 20 μm).

Article Snippet: Purified viruses were titered using a qRT-PCR based method to determine the number of DNAse resistant viral particles and the results were reported as genomes copies (GC)/mL and this was performed as previously described [ ] using PCR primers specific for GluN2A ( Life Technologies , CatRn00561341_m1), GluN2B ( Life Technologies , Cat# Mm00433820_m1) and GFP ( Life technologies , Cat# Mr04329676_mr).

Techniques: Expressing, In Vitro, Plasmid Preparation, Transfection, Fluorescence, Microscopy, Staining

Journal: Molecular Brain

Article Title: The production of viral vectors designed to express large and difficult to express transgenes within neurons

doi: 10.1186/s13041-015-0100-7

Figure Lengend Snippet: GluN2 and GFP transgene expression in vitro mediated by lentivirus. In this experiment, 293FT cells were transduced with lentiviruses designed to express GFP, Flag-GluN2A, or Flag-GluN2B from a CMV or a TRE3G promoter as indicated. TRE3G promoter containing viruses were transduced into cells that were also transfected with the pTet-Off plasmid. Forty eight hours after viral transduction, native GFP expression was observed via fluorescence microscopy and Flag-GluN2 expression was observed via ICC and fluorescence microscopy. Images depict DAPI stained nuclei with the same fields viewed for GFP or Flag-GluN2 (Texas Red) transgene expression. Non-transduced cells and cells transfected with pRK5-Flag-GluN2 plasmids were processed as negative and positive ICC controls respectively. These viruses were capable of conferring GluN2 or GFP transgene expression as intended.

Article Snippet: Purified viruses were titered using a qRT-PCR based method to determine the number of DNAse resistant viral particles and the results were reported as genomes copies (GC)/mL and this was performed as previously described [ ] using PCR primers specific for GluN2A ( Life Technologies , CatRn00561341_m1), GluN2B ( Life Technologies , Cat# Mm00433820_m1) and GFP ( Life technologies , Cat# Mr04329676_mr).

Techniques: Expressing, In Vitro, Transduction, Transfection, Plasmid Preparation, Fluorescence, Microscopy, Staining

Journal: Molecular Brain

Article Title: The production of viral vectors designed to express large and difficult to express transgenes within neurons

doi: 10.1186/s13041-015-0100-7

Figure Lengend Snippet: GluN2 and GFP transgene expression in vivo mediated by lentivirus. In this experiment lentiviruses designed to express Flag-GluN2A, Flag-GluN2B, or GFP under the control of 0.5Synapsin, CMV, 0.4αCaMKII, or 1.3αCaMKII promoters as indicated were infused into rat basal and lateral amygdala nuclei (BLA). Lentiviruses designed to express Flag-GluN2A, Flag-GluN2B, or GFP under the control of a TRE3G promoter were infused into αCaMKII-tTA transgenic mice. Ten days following viral infusion, coronal sections were prepared that contained the BLA and native GFP expression was observed via fluorescence microscopy and Flag-GluN2 expression was observed via immunohistochemistry, (IHC) and fluorescence microscopy. Images depict DAPI stained nuclei with the same fields viewed for GFP or Flag-GluN2 (Texas Red) transgene expression. Lentiviruses designed to express GFP from 0.5Synapsin, 0.4αCaMKII, 1.3αCaMKII, TRE3G promoters were capable of conferring GFP expression which was localized to neurons. Lentivirus designed to express GFP from a CMV promoter primarily conferred expression of GFP within glia cells. Lentiviruses designed to express Flag-GluN2A/B from either a 0.4αCaMKII or 1.3αCaMKII promoter were not capable of conferring GluN2 expression. Lentiviruses designed to express Flag-GluN2A/B from a TRE3G promoter were capable of conferring GluN2 expression as determined by IHC. Coronal sections from naïve controls were processed as a negative control for anti-Flag IHC (Negative control = coronal rat section and Negative control * = coronal mouse section), (scale bar = 50 μm).

Article Snippet: Purified viruses were titered using a qRT-PCR based method to determine the number of DNAse resistant viral particles and the results were reported as genomes copies (GC)/mL and this was performed as previously described [ ] using PCR primers specific for GluN2A ( Life Technologies , CatRn00561341_m1), GluN2B ( Life Technologies , Cat# Mm00433820_m1) and GFP ( Life technologies , Cat# Mr04329676_mr).

Techniques: Expressing, In Vivo, Control, Transgenic Assay, Fluorescence, Microscopy, Immunohistochemistry, Staining, Negative Control

Journal: Molecular Brain

Article Title: The production of viral vectors designed to express large and difficult to express transgenes within neurons

doi: 10.1186/s13041-015-0100-7

Figure Lengend Snippet: Lentivirus designed with a 1.3 αCaMKII promoter and an intron conferred in vitro and in vivo expression of full length GluN2A. (A) Viral genome maps for pLenti7.3 vectors containing either the Flag-GluN2A coding region or a MCS with a 1.3αCaMKII promoter and intron. (B) A lentivirus plasmid designed to express Flag-GluN2A that included an intron from a 1.3αCaMKII promoter was transfected into N2A cells and 24 hours post transfection the cells were examined by ICC for Flag-GluN2 expression. This plasmid was not capable of conferring Flag-GluN2A expression. However when this plasmid was linearized, (Lenti-1.3αCaMKII + intron-GluN2A*) and then transfected as above to eliminate the RSV promoter from interfering with GluN2 expression, Flag-GluN2 expression was indeed detected. Non-transfected cells and cells transfected with the pRK5-Flag-GluN2A plasmid were processed as negative and positive ICC controls respectively (scale bar = 20 μm). (C) A lentivirus designed to express Flag-GluN2A that included an intron from a 1.3αCaMKII promoter was infused into the rat BLA. Ten days following viral infusion, coronal sections were prepared that contained the BLA and Flag-GluN2 expression was observed via IHC and fluorescence microscopy. Coronal sections from non-infused animals were processed as a negative control for Flag-IHC. (D) In this experiment, it was determined if the Flag-GluN2A expression from the 1.3αCaMKII-intron virus was predominantly restricted to neurons, by performing an IHC for Flag-GluN2A and the neuronal marker NeuN. Images depict Flag-GluN2A staining (Tx-red) with the same fields viewed for NeuN (FITC) staining and a merged image of these two images. This virus was capable of conferring GluN2A expression predominantly within neurons. Arrows point to a subset of NeuN positive cells that are also Flag-GluN2A positive (E) Quantification of data presented in (D), error bars represent the standard error of the mean, (scale bar = 50 μm).

Article Snippet: Purified viruses were titered using a qRT-PCR based method to determine the number of DNAse resistant viral particles and the results were reported as genomes copies (GC)/mL and this was performed as previously described [ ] using PCR primers specific for GluN2A ( Life Technologies , CatRn00561341_m1), GluN2B ( Life Technologies , Cat# Mm00433820_m1) and GFP ( Life technologies , Cat# Mr04329676_mr).

Techniques: In Vitro, In Vivo, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy, Negative Control, Virus, Marker, Staining

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 < 0.05 by Student unpaired t test.

Article Snippet: Some paraffin sections were immunostained with antibodies against various NMDA receptor subunits (anti-NR1 from Abcam, anti-NR2A and anti-NR2B from Thermo Fisher Scientific, and anti-NR2C from Alomone Labs).

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

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 < 0.05 by Student unpaired t test.

Article Snippet: Some paraffin sections were immunostained with antibodies against various NMDA receptor subunits (anti-NR1 from Abcam, anti-NR2A and anti-NR2B from Thermo Fisher Scientific, and anti-NR2C from Alomone Labs).

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

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 < 0.05 by Student unpaired t test.

Article Snippet: Some paraffin sections were immunostained with antibodies against various NMDA receptor subunits (anti-NR1 from Abcam, anti-NR2A and anti-NR2B from Thermo Fisher Scientific, and anti-NR2C from Alomone Labs).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

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 < 0.05 by Student unpaired t test.

Article Snippet: Some paraffin sections were immunostained with antibodies against various NMDA receptor subunits (anti-NR1 from Abcam, anti-NR2A and anti-NR2B from Thermo Fisher Scientific, and anti-NR2C from Alomone Labs).

Techniques: Immunohistochemistry, Negative Control, Staining

Journal: Molecular Neurobiology

Article Title: Leptin Contributes to Neuropathic Pain via Extrasynaptic NMDAR-nNOS Activation

doi: 10.1007/s12035-020-02180-1

Figure Lengend Snippet: NR2A and NR2B antagonists prevented and reversed mechanical allodynia in rats with SNI. a The threshold force of SNI-induced mechanical allodynia on the ipsilateral hind paw was significantly decreased on day 7 and continued until day 14. Intrathecal treatment with the NR2A-selective antagonist NVP-AAM077 (4 nmol) and the NR2B-selective antagonist Ro25-6981 (20 nmol) once daily for 14 days prevented the development of mechanical allodynia on the hind paw ipsilateral to SNI on days 3, 5, 7, 10, and 14. c A single intrathecal administration of NVP-AAM077 (4 nmol) and Ro25-6981 (20 nmol) on day 14 attenuated mechanical allodynia at 30 min after the treatment, and the effect of NVP-AAM077 was maintained for 24 h. b , d NVP-AAM077 and Ro25-6981 did not change the mechanical nociceptive threshold of the contralateral hind paw in the same rat. e A single intrathecal administration of 10 nmol MK-801 on day 14 reversed SNI-induced mechanical allodynia 30 min after the treatment. NVP, NVP-AAM077; Ro25, Ro25-6981; MK, MK-801. Data are shown as the means ± SE. * P < 0.05, ** P < 0.01 versus vehicle. # P < 0.05, ## P < 0.01 versus before the single intrathecal treatment

Article Snippet: Membranes were blocked with 5% nonfat dried milk and incubated overnight (4 °C) with anti-NR2A antibody (BOSTER, China: 1:400, rabbit polyclonal), anti-NR2B antibody (Abcam, USA: 1:1000, rabbit polyclonal) [ ] and anti-nNOS antibody (BD Biosciences, USA: 1:1000, mouse monoclonal).

Techniques:

Journal: Molecular Neurobiology

Article Title: Leptin Contributes to Neuropathic Pain via Extrasynaptic NMDAR-nNOS Activation

doi: 10.1007/s12035-020-02180-1

Figure Lengend Snippet: NR2A and NR2B antagonists prevented exogenous leptin-induced mechanical allodynia. a Intrathecal leptin (50 μg) treatment in naïve rats, given once daily for 7 days, induced mechanical allodynia on day 7. Coadministration of leptin with 4 nmol NVP-AAM077 or 20 nmol Ro25-6981 attenuated the behavioral changes ( n = 5). b NVP-AAM077 and Ro25-6981 alone did not change the baseline nociceptive threshold ( n = 6). lep, leptin; NVP, NVP-AAM077; Ro25, Ro25-6981. Data are shown as the means ± SE. ** P < 0.01 versus day 0

Article Snippet: Membranes were blocked with 5% nonfat dried milk and incubated overnight (4 °C) with anti-NR2A antibody (BOSTER, China: 1:400, rabbit polyclonal), anti-NR2B antibody (Abcam, USA: 1:1000, rabbit polyclonal) [ ] and anti-nNOS antibody (BD Biosciences, USA: 1:1000, mouse monoclonal).

Techniques:

Journal: Molecular Neurobiology

Article Title: Leptin Contributes to Neuropathic Pain via Extrasynaptic NMDAR-nNOS Activation

doi: 10.1007/s12035-020-02180-1

Figure Lengend Snippet: Leptin enhancement of NR2B- but not NR2A-mediated currents in dissociated lamina II neurons in naïve rats. a Treatment with the NR2A-selective antagonist NVP-AAM077 (0.4 μM) plus the NR2B-selective antagonist Ro25-6981 (1 μM) blocked NMDAR-mediated currents ( n = 8). b Exposure to leptin (100 nM) for 5 min did not change NMDAR-mediated currents after blockade with Ro25-6981 (1 μM) ( n = 10). c Exposure to leptin (100 nM) for 5 min enhanced NMDAR-mediated currents after inhibition by 0.4 μM NVP-AAM077 ( n = 9). d Histograms showing the effect of leptin on NMDAR-mediated currents after inhibition by NVP-AAM077 or Ro25-6981. Data are shown as the means ± SE. lep, leptin; NVP, NVP-AAM077; Ro, Ro25-6981. * P < 0.05, ** P < 0.01 vs. vehicle; # P < 0.05 vs NVP

Article Snippet: Membranes were blocked with 5% nonfat dried milk and incubated overnight (4 °C) with anti-NR2A antibody (BOSTER, China: 1:400, rabbit polyclonal), anti-NR2B antibody (Abcam, USA: 1:1000, rabbit polyclonal) [ ] and anti-nNOS antibody (BD Biosciences, USA: 1:1000, mouse monoclonal).

Techniques: Inhibition

Journal: Molecular Neurobiology

Article Title: Leptin Contributes to Neuropathic Pain via Extrasynaptic NMDAR-nNOS Activation

doi: 10.1007/s12035-020-02180-1

Figure Lengend Snippet: Leptin enhancement of NR2B, but not NR2A, expression in cultured DRG neurons. a Immunohistochemistry results showed that administration of leptin in culture medium for 72 h upregulated NR2B expression in a dose-dependent manner (2 ng/ml leptin had the maximal enhancement effect), and cotreatment with 1 μM Ro25-6981 diminished the upregulation. b Leptin at 2 ng/ml slightly enhanced NR2A expression, which was attenuated by 0.4 μM NVP-AAM077. c – f Western blot results showed that administration of leptin (2 ng/ml) to culture medium for 72 h significantly upregulated NR2B expression ( c , d ) but not NR2A expression ( e , f ) in cultured DRG neurons. The NR2B upregulation was blocked by 1 μM Ro25-6981 ( c , d ). Neither 1 μM Ro25-6981 nor 0.4 μM NVP-AAM077 alone changed the baseline expression of NR2B or NR2A. lep, leptin; NVP, NVP-AAM077; Ro, Ro25-6981. n = 3. Scale bar, 50 μm. * P < 0.05 vs vehicle

Article Snippet: Membranes were blocked with 5% nonfat dried milk and incubated overnight (4 °C) with anti-NR2A antibody (BOSTER, China: 1:400, rabbit polyclonal), anti-NR2B antibody (Abcam, USA: 1:1000, rabbit polyclonal) [ ] and anti-nNOS antibody (BD Biosciences, USA: 1:1000, mouse monoclonal).

Techniques: Expressing, Cell Culture, Immunohistochemistry, Western Blot

Journal: Molecular Neurobiology

Article Title: Leptin Contributes to Neuropathic Pain via Extrasynaptic NMDAR-nNOS Activation

doi: 10.1007/s12035-020-02180-1

Figure Lengend Snippet: Leptin-mediated enhancement of nNOS expression was blocked by an NR2B antagonist. Immunohistochemistry ( a ) and Western blot ( b and c ) results showed that administration of leptin (2 ng/ml) to culture medium for 72 h significantly upregulated nNOS expression in cultured DRG neurons. The upregulation of nNOS expression by leptin was significantly prevented by coapplication of the NR2B antagonist Ro25-6981 (1 μM) and slightly attenuated by the NR2A antagonist NVP-AAM077 (0.4 μM). Ro25-6981 (1 μM) and NVP-AAM077 (0.4 μM) alone did not change baseline nNOS expression. Lep, leptin; NVP, NVP-AAM077; Ro, Ro25-6981. n = 3. Scale bar, 50 μm. ** P < 0.01 vs vehicle; # P < 0.05 vs leptin

Article Snippet: Membranes were blocked with 5% nonfat dried milk and incubated overnight (4 °C) with anti-NR2A antibody (BOSTER, China: 1:400, rabbit polyclonal), anti-NR2B antibody (Abcam, USA: 1:1000, rabbit polyclonal) [ ] and anti-nNOS antibody (BD Biosciences, USA: 1:1000, mouse monoclonal).

Techniques: Expressing, Immunohistochemistry, Western Blot, Cell Culture

Journal: Nature chemical biology

Article Title: Identification and characterization of PPARα ligands in the hippocampus

doi: 10.1038/nchembio.2204

Figure Lengend Snippet: A) Heat map analysis shows the PCR-based microarray analysis of plasticity-associated genes in the hippocampus of WT and αKO ( Ppara -null) mice. Three mice were used in each group. B) Venn diagram of plasticity-associated genes shows the number of genes inhibited (28; red circle), stimulated (34; green circle) and unchanged (22; overlapped region) in Ppara -null hippocampus. C) Real-time PCR analyses of Arc, Creb, Grin2a, Grin2b, and Gria1 mRNAs were performed to confirm the array results. Results are mean ± SEM of three mice. a p<0.001 vs WT . D) Hippocampal tissue of 6- to 8-week-old WT (n=3) and Ppara -null (n=3) mice were immunostained for MAP-2 (green) and PSD-95 (red). The representative image was taken from CA1 region of the hippocampus. Scale bar = 10μm. E) The magnified view of region enclosed in the box is shown in the image. Scale bar = 10 μm. Results represent analysis of three hippocampal sections of each of three mice per group. The expression of NR-2A, GluR1, PSD95, Arc, and CREB in hippocampal tissue of WT ( n=3 ) and Ppara -null ( n=3 ) mice was further assessed by Western blot (F) followed by densitometric analyses (G) after normalizing with actin. For raw uncut blots, please see . Results are mean ± SEM of three mice. a p<0.001 vs WT .

Article Snippet: Rabbit polyclonal anti-PPARα antibody (Abcam; Cat# ab189159; WB and IHC), mouse anti-NeuN antibody (Millipore; Cat# MAB377), rabbit polyclonal anti-PPARβ antibody (Abcam; Cat # ab8937; WB and IHC), anti-PPARγ antibody (Abcam; Cat# ab66343; WB and IHC), anti-NMDAR2A antibody (Cell Signaling for WB at a dilution of 1:1000, Cat #4205; Abcam for IHC, Cat# ab169873), anti-GluR1 antibody (Cell Signaling for WB at a dilution of 1:1000, Cat #13185; Abcam for IHC, Cat # ab131507), anti-CREB antibody ( Cell Signaling for WB at a dilution of 1:1000 and IC at a dilution of 1:200, Cat# 9104), and anti-Arc antibody (Abcam for WB at a dilution of 1:1000, Cat # ab118929) were used in this study.

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

Journal: Brain research

Article Title: Antibody-mediated targeted gene transfer of helper virus-free HSV-1 vectors to rat neocortical neurons that contain either NMDA receptor 2B or 2A subunits

doi: 10.1016/j.brainres.2011.08.010

Figure Lengend Snippet: The numbers of expressing cells and the percentage of NR2B-IR or NR2A-IR costaining for rats sacrificed at 8 days after co-injection of either pINS-TH-NFHlac/gC--ZZ+anti-NR2B or +anti-NR2A and pINS-TH-NFHflag-TH/ires/aadc/gC--wt into POR cortex

Article Snippet: The following antibodies were used to target gene transfer to, or detect, specific NMDA receptor subunits: Rabbit anti-NMDA NR2B (Sigma, M-265, for targeted gene transfer), rabbit anti-NMDA NR2B (Santa Cruz Biotechnology, sc-9057, for immunohistochemistry), rabbit anti-NMDA NR2A (Santa Cruz Biotechnology, sc-9056, for targeted gene transfer), and rabbit anti-NMDA NR2A (Invitrogen, A-6473, for immunohistochemistry).

Techniques: Expressing

Journal: Brain research

Article Title: Antibody-mediated targeted gene transfer of helper virus-free HSV-1 vectors to rat neocortical neurons that contain either NMDA receptor 2B or 2A subunits

doi: 10.1016/j.brainres.2011.08.010

Figure Lengend Snippet: Costaining for recombinant expression in NMDA NR2A-containing neurons in a rat sacrificed at eight days after co-injection of pINS-TH-NFHlac/gC--ZZ+anti-NR2A and pINS-TH-NFHflag-TH/ires/aadc/gC--wt into POR cortex. Sections were costained using either mouse anti-β-gal or mouse anti-flag and rabbit anti-NMDA NR2A. (A–C) pINS-TH-NFHlac/gC--ZZ+anti-NR2A supports β-gal expression predominantly in NR2A-containing neurons; β-gal-IR (A), NR2A-IR (B), and merge (C). Arrows, costained cells. (D–F) pINS-TH-NFHflag-TH/ires/aadc/gC--wt supports TH expression predominantly in neurons that lack NR2A-IR; flag-IR (D), NR2A-IR (E), and merge (F). Arrowheads, flag-IR only. Scale bar: 30 μm.

Article Snippet: The following antibodies were used to target gene transfer to, or detect, specific NMDA receptor subunits: Rabbit anti-NMDA NR2B (Sigma, M-265, for targeted gene transfer), rabbit anti-NMDA NR2B (Santa Cruz Biotechnology, sc-9057, for immunohistochemistry), rabbit anti-NMDA NR2A (Santa Cruz Biotechnology, sc-9056, for targeted gene transfer), and rabbit anti-NMDA NR2A (Invitrogen, A-6473, for immunohistochemistry).

Techniques: Recombinant, Expressing, Injection

Journal: Molecular Neurobiology

Article Title: Ouabain Modulates the Functional Interaction Between Na,K-ATPase and NMDA Receptor

doi: 10.1007/s12035-020-01984-5

Figure Lengend Snippet: Proximity ligation assay (PLA) images of rat hippocampal neurons. PLA was performed using antibodies against Na,K-ATPase α-subunits (NKAα1 and NKAα3) and against GluN2-subunits (GluN2A and GluN2B). Omission of primary antibodies was used as negative control, and antibodies against GluN1 was used as a positive control. Red dots indicate PLA signal; cell nuclei are identified using DAPI stain. Upper left image is PLA of NKAα3 with GluN2B counterstained with pan neuronal marker in green to visualize neurons with extensions. Scale bar = 50 μm

Article Snippet: Primary antibodies used in this study were mouse anti-Na,K-ATPase α1 (5.7 μg/ml, DHSB), mouse anti-Na,K-ATPase α3 (1 μg/ml, Thermo Fisher Scientific), mouse anti-GluN1 (1:1000, Millipore), rabbit anti-GluN2A (2 μg/ml for immunocytochemistry, Millipore), rabbit anti-GluN2B (2 μg/ml, Millipore), mouse anti-GluN2B (2.5 μg/ml for immunocytochemistry), rabbit anti-GluN2B-pY1252/1336/1472 (1:100, Phosphosolutions), rabbit anti-GFP (4 μg/ml, Thermo Fischer Scientific), and mouse anti-Pan neuronal marker (1:1000, Millipore).

Techniques: Proximity Ligation Assay, Negative Control, Positive Control, Staining, Marker

Journal: Molecular Neurobiology

Article Title: Ouabain Modulates the Functional Interaction Between Na,K-ATPase and NMDA Receptor

doi: 10.1007/s12035-020-01984-5

Figure Lengend Snippet: Super-resolution imaging shows that NMDAR and Na+,K + -ATPase are in close proximity in hippocampal neurons. a – d Overview images of dendritic segments from dSTORM experiments with antibody labeled GluN2-subunits (GluN2A and GluN2B) and Na,K-ATPase α-subunits (NKAα1 and NKAα3). The GluN2 antibodies were tagged with Alexa-647 conjugated secondary antibodies, and Na,K-ATPase antibodies were tagged with Atto-488 conjugated secondary antibodies. Images show Gaussian representations of clusters containing single localized molecules of dendritic segments and GluN2 subunit containing clusters. Cumulative probability distributions of quantified distances from GluN2 subunits to the closest Na,K-ATPase α and vice versa. Histogram insets show the relative frequency of distances from GluN2 subunits to the closest Na,K-ATPase α (insets). Scale bar = 5 μm (overviews of dendrites) and 200 nm (zoom in on clusters)

Article Snippet: Primary antibodies used in this study were mouse anti-Na,K-ATPase α1 (5.7 μg/ml, DHSB), mouse anti-Na,K-ATPase α3 (1 μg/ml, Thermo Fisher Scientific), mouse anti-GluN1 (1:1000, Millipore), rabbit anti-GluN2A (2 μg/ml for immunocytochemistry, Millipore), rabbit anti-GluN2B (2 μg/ml, Millipore), mouse anti-GluN2B (2.5 μg/ml for immunocytochemistry), rabbit anti-GluN2B-pY1252/1336/1472 (1:100, Phosphosolutions), rabbit anti-GFP (4 μg/ml, Thermo Fischer Scientific), and mouse anti-Pan neuronal marker (1:1000, Millipore).

Techniques: Imaging, Labeling