Journal: Antioxidants (Basel, Switzerland)

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

doi: 10.3390/antiox11081471

Figure Lengend Snippet: Figure 2. (A) The chemical structure of ergotamine. (B) The H2O-injected oocytes did not cause any change with the treatment of 100 µM glutamate (n = 6–8 oocytes from four different frogs). (C–F) Glutamate induced inward currents with or without ergotamine (30 µM and 10 µM). For each subunit of NMDARs, the responses after treating with either glutamate (100 µM) alone or together with ergotamine (30 and 10 µM). Voltage clamp recording was conducted at a holding potential of −80 mV. The coapplication of ergotamine with glutamate resulted in modulation of the recombinant receptors (C) NR1a/NR2A, (D) NR1a/NR2B, (E) NR1a/NR2C, and (F) NR1a/NR2D, which in turn reduced glutamate-evoked inward current in a reversible manner (n = 6–8 oocytes from four different frogs).

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

Techniques: Injection, Recombinant

Journal: Antioxidants (Basel, Switzerland)

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

doi: 10.3390/antiox11081471

Figure Lengend Snippet: Figure 3. Computational molecular modeling of ergotamine docked to the human NR1a/NR2A receptor. (A,C) Side views of the docked ergotamine complex with NMDA channel. (B,D) Binding pocket and docking results of ergotamine and NMDA channel, respectively.

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

Techniques: Binding Assay

Journal: Antioxidants (Basel, Switzerland)

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

doi: 10.3390/antiox11081471

Figure Lengend Snippet: Figure 4. Predicted binding mode of ergotamine and all the favorable interactions with several residues in the active site of the human NR1a/NR2A receptor. (A,B) Interaction between ergo- tamine and wild-type NR1a/NR2A. (C) Residues in wild-type NR1a/NR2A receptor interacting with the ergotamine molecule. (D) Change in the interaction distance of ergotamine in mutant-type NR1a/NR2A receptor. Based on the change in this distance, the residue that directly interacts with ergotamine was identified.

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

Techniques: Binding Assay, Mutagenesis, Residue

Journal: Antioxidants (Basel, Switzerland)

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

doi: 10.3390/antiox11081471

Figure Lengend Snippet: Figure 5. The inward current of several mutant types on the glutamate-evoked current of NR1a/NR2A receptor with or without ergotamine. Representative traces of current induced by application of glutamate (100 µM) alone or together with ergotamine (100 µM) for various mutants: (A) mutant 1 (NR1a subunit W167A and NR2A wild-type), (B) mutant 2 (NR1a subunit H168A and NR2A wild-type), (C) mutant 3 (NR1a subunit V169A and NR2A wild-type), (D) mutant 4 (NR1a wild-type and NR2A subunit P435A), (E) mutant 5 (NR1a wild-type and NR2A subunit N466A), and (F) mutant 6 (double mutant-type NR1a subunit V169A and NR2A subunit N466A). Experiments were performed separately, and data were collected from several oocytes (n = 6–8 oocytes from four different frogs).

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

Techniques: Mutagenesis

Journal: Scientific reports

Article Title: Epilepsy-associated GRIN2A mutations reduce NMDA receptor trafficking and agonist potency - molecular profiling and functional rescue.

doi: 10.1038/s41598-017-00115-w

Figure Lengend Snippet: Figure 1. CADD scores and protein modelling predict stronger functional effects for EAS-associated GRIN2A mutations than in controls. (a) Protein structure model of NMDAR (PDB ID 4TLL): GluN1 (grey and green), GluN2 (B in this structure) (blue and red). Membrane would be horizontal in this image with the NTD and ABD in the extracellular space. Intracellular C-Terminal domain would be below the transmembrane domain (not present in this structure). (b) Schematic linear representation of GluN2A with the domains annotated. Black rectangles indicate transmembrane domains. Plot of scaled CADD scores against GluN2A amino acid position for missense variants. Black dots represent scores for 65/6474 individuals from the Exome Variant Server (EVS) that had missense variants in GRIN2A and coloured symbols are scores for variants found in individuals with EAS disorders. The horizontal dotted line indicates the scaled CADD score cut off of 20 for a highly likely deleterious variant. (c) Protein structure model of the NTD of NMDAR (PDB ID 3QEL): GluN1 (grey), GluN2 (B in this structure) (red). Mutations considered in this domain highlighted (conserved between GluN2A and B). (d) Protein structure model of the LBD of NMDAR (PDB ID 2A5T): GluN1 (grey), GluN2A (blue). Mutations considered in this paper highlighted, as well as agonists.

Article Snippet: Human GRIN2A (GenBank accession: NM_000833.3) cDNA clone was purchased from OriGene Technologies (Cat#: SC122120).

Techniques: Functional Assay, Membrane, Variant Assay

Journal: Scientific reports

Article Title: Epilepsy-associated GRIN2A mutations reduce NMDA receptor trafficking and agonist potency - molecular profiling and functional rescue.

doi: 10.1038/s41598-017-00115-w

Figure Lengend Snippet: Figure 2. Selected GRIN2A mutations protect against glutamate-induced toxicity in HEK cells. (a) Superimposed bright field and pseudocolour red images, indicating fluorescent Cytotox Red dye in dead cells, of HEK cells transiently transfected with various GRIN2A mutant constructs, or empty vector. Free glutamate and glycine in the culture media causes cell death in those expressing functional NMDARs over 48 hours. Images captures at 20x using the IncuCyte live-cell imaging system. (b) Representative time course of cell death, normalised to initial confluency in each well, n = 5 wells, Mean ± SEM. (c) Plot of cell mortality for GRIN2A mutants normalised to initial confluency per well. Mutants P79R, C231Y, C436R, G483R, M705V, D731N and I814T are protective against glutamate toxicity either due to reduced trafficking and/or reduced functionality of the receptors. ***p < 0.001 Dunnett’s corrected one-way ANOVA. Averaged data from n = 15 wells per construct except for G483R, E714K, D933N and N976S where n = 12 wells, 3 × 104 cells/well. Mean ± SEM.

Article Snippet: Human GRIN2A (GenBank accession: NM_000833.3) cDNA clone was purchased from OriGene Technologies (Cat#: SC122120).

Techniques: Transfection, Mutagenesis, Construct, Plasmid Preparation, Expressing, Functional Assay, Live Cell Imaging

Journal: Scientific reports

Article Title: Epilepsy-associated GRIN2A mutations reduce NMDA receptor trafficking and agonist potency - molecular profiling and functional rescue.

doi: 10.1038/s41598-017-00115-w

Figure Lengend Snippet: Figure 3. GRIN2A mutations alter the response to glutamate and glycine. (a,b) Pseudocolour images and representative single-cell traces from calcium-flux imaging for HEK cells transfected with either WT or G483R GRIN2A showing different calcium responses caused by the application of increasing concentrations of glutamate (30 nM–30 µM red arrows). Individual cell traces in cyan and the mean response in red. (c,d) Normalised concentration response curves (CRCs) to increasing concentrations of glutamate from single cell calcium-flux imaging. In (c) 4 mutants; P79R, C231Y, C483R and M705V have reduced agonist potency, while C436R and D731N show no response to glutamate. Four mutant constructs (d), show an unaltered response to glutamate. (e,f) Normalised CRCs to increasing glycine concentration with constant 3 µM glutamate – the mutations show similar responses as to glutamate. See Table 2 for n to create averaged data per construct, 3 × 104 cells/well. Error bars ± SEM. (g) Representative continuous voltage-clamp recordings obtained from HEK cells transfected with WT or G483R GRIN2A plasmid. Bars above the recording indicate glutamate application (co- applied with 30 µM glycine). Application of increasing concentrations of glutamate shows a progressive increase in current observed, the sensitivity of which is shifted to higher concentrations of glutamate in the G483R mutant compared to WT. (h) Normalised CRC to glutamate as recorded in (g) indicates the same response as for single cell calcium imaging (n = 3).

Article Snippet: Human GRIN2A (GenBank accession: NM_000833.3) cDNA clone was purchased from OriGene Technologies (Cat#: SC122120).

Techniques: Imaging, Transfection, Concentration Assay, Mutagenesis, Construct, Plasmid Preparation

Journal: Scientific reports

Article Title: Epilepsy-associated GRIN2A mutations reduce NMDA receptor trafficking and agonist potency - molecular profiling and functional rescue.

doi: 10.1038/s41598-017-00115-w

Figure Lengend Snippet: Figure 4. GRIN2A mutations alter the number of cells responding to glutamate. (a–d) Each panel contains pseudocolour images of HEK cells transfected with WT or mutant GRIN2A showing co-localisation of responses to application of 100 µM glutamate +30 µM glycine (green - activation of surface-expressing GluN2A receptors) and subsequently 100 µM MgATP (red - activation of endogenous P2Y receptors). Panels also show single-cell traces from the same experiment, indicating the different effects of these agonists on cells transfected with (a) WT, (b) M705V, (c) C231Y or (d) C436R mutants. Individual cell traces displayed in cyan and the mean response shown in red. (e) Ratio of the number of transfected HEK cells responding to 100 µM glutamate +30 µM glycine subsequent to 100 µM MgATP. Dunnett’s corrected one-way ANOVA compared to WT, **p < 0.01, ***p < 0.001, ns = non-significant. Data averaged from n = 12 wells, 3 × 104 cells/well, for each construct over 2 assays. Error bars ± SEM.

Article Snippet: Human GRIN2A (GenBank accession: NM_000833.3) cDNA clone was purchased from OriGene Technologies (Cat#: SC122120).

Techniques: Transfection, Mutagenesis, Activation Assay, Expressing, Construct

Journal: Scientific reports

Article Title: Epilepsy-associated GRIN2A mutations reduce NMDA receptor trafficking and agonist potency - molecular profiling and functional rescue.

doi: 10.1038/s41598-017-00115-w

Figure Lengend Snippet: Figure 5. GRIN2A mutations reduce total protein levels and membrane trafficking of GluN2A. (a) Representative Western blot of HEK lysates probed with anti-GluN2A antibody (top), and anti-GAPDH (bottom) as a loading control. Bands around 180 kDa indicate GluN2A. WT, wild type, UT – untransfected. Right is Amersham Full-Range rainbow molecular weight marker with the blot imaged in visible light. (b) Plot of amount of GluN2A protein, normalised to WT, from Western blotting of total cell lysates of transiently co- transfected HEK cells 48-hours post transfection. Average of 3 blots from 3 independent transfections. Error bars indicated SEM. (c) Fixed and immunolabelled co-transfected HEK cells with anti-HA antibody (red) to detect surface GluN2A expression, and fixed, permeabilised and immunolabelled with the same antibody to detect total GluN2A protein levels. Scale bar 25 µm. Nuclei stained with Hoechst (blue). (d) Quantitation of surface and total GluN2A protein levels, averaged over the total number of cells analyzed for each condition (n is between 903 to 2255 cells), reveals greatly reduced surface expression of GluN2A as measured by fluorescence intensity. Dunnett’s corrected one-way ANOVA for membrane or total intensity as compared to WT *p < 0.05, **p < 0.01, ***p < 0.001, ns = non-significant. Average ± SEM. (e) Relative surface levels of NMDARs correlated with the log of glutamate EC50 (Pearson’s coefficient of determination r2 = 0.77, two-tailed p = 0.002). (f) Normalised CRC from single-cell calcium-flux imaging. Response to increasing concentrations of glutamate from HEK cells co-transfected with decreasing quantities of WT GRIN2A per well (100% = 320 ng) with standard 320 ng GRIN1 per well. Response is compared to mutant P79R. Error bars ± SEM.

Article Snippet: Human GRIN2A (GenBank accession: NM_000833.3) cDNA clone was purchased from OriGene Technologies (Cat#: SC122120).

Techniques: Membrane, Western Blot, Control, Molecular Weight, Marker, Transfection, Expressing, Staining, Quantitation Assay, Fluorescence, Two Tailed Test, Imaging, Mutagenesis

Journal: Scientific reports

Article Title: Epilepsy-associated GRIN2A mutations reduce NMDA receptor trafficking and agonist potency - molecular profiling and functional rescue.

doi: 10.1038/s41598-017-00115-w

Figure Lengend Snippet: Figure 6. Pharmacological rescue of functional deficits can be achieved for selected GRIN2A mutants. Examples of single-cell imaging of HEK transfected with (a) WT and (b) C231Y mutant showing calcium- influx response to 300 nM glutamate before and after incubation with 1 µM PAM and in comparison to maximal 30 µM glutamate. (c) Graph of single cell imaging data showing response of WT and mutants P79R, C231Y and G483R to 300 nM glutamate with and without 1 µM PAM. Bonferroni corrected ANOVA ***p < 0.001, n = 15 wells (over 3 assays) for each construct, 3 × 104 cells/well. Mean ± SEM. (d) Examples of single cell imaging of HEK transfected with C231Y mutant showing calcium-influx response to increasing concentrations of glutamate (30 nM, 100 nM, 300 nM, 1 µM, 10 µM and 30 µM arrows) top, and below, increasing concentrations of glutamate (30 nM, 100 nM, 300 nM, 1 µM and 30 µM arrows) with constant 1 µM PAM. Individual cell traces displayed in cyan and the mean response shown in red. (e–h) Concentration response curves of mutant (P79R, C231Y, G483R or M705V) GRIN2A transfected in HEK cells to increasing concentrations of glutamate (30 nM to 30 µM) during incubation with 1 µM PAM recorded from single-cell calcium-flux imaging. Each graph shows the response of the WT GRIN2A construct with no PAM in red (curve from Fig. 3c) and the response of the mutant before (solid line) and after (dashed line) the addition of PAM. p < 0.0001 for LogEC50 for each construct + PAM when compared to without PAM addition. Data averaged from n = 12 wells for each construct, 3 × 104 cells/well, (over 3 assays). Error bars ± SEM.

Article Snippet: Human GRIN2A (GenBank accession: NM_000833.3) cDNA clone was purchased from OriGene Technologies (Cat#: SC122120).

Techniques: Functional Assay, Imaging, Transfection, Mutagenesis, Incubation, Comparison, Construct, Concentration Assay

Journal: PLoS ONE

Article Title: MPX-004 and MPX-007: New Pharmacological Tools to Study the Physiology of NMDA Receptors Containing the GluN2A Subunit

doi: 10.1371/journal.pone.0148129

Figure Lengend Snippet: The values are IC 50 s for inhibition of Ca 2+ responses mediated by GluN2A receptors expressed in HEK cells.

Article Snippet: The human GRIN1 gene transcript variant NR1-3 (Origene, SKU#: SC115601, Rockville, MD), GRIN2A gene (Origene, SKU#: SC326381), GRIN2B gene (B’SYS, Witterswil, Switzerland), GRIN2C gene (NCBI locus NM_000835, optimized for oocyte expression and synthesized by GenScript, Piscataway, NJ) and GRIN2D gene (NCBI locus NM_000836, sequence optimized and synthesized by GenScript) were linearized and transcribed using the mMessage mMachine kit (Life Technologies) and promoters SP6 (for GluN1) and T7 (for GluN2A-2D). cRNAs were mixed together at a ratio of 1 GluN1:2 GluN2.

Techniques: Inhibition

Journal: PLoS ONE

Article Title: MPX-004 and MPX-007: New Pharmacological Tools to Study the Physiology of NMDA Receptors Containing the GluN2A Subunit

doi: 10.1371/journal.pone.0148129

Figure Lengend Snippet: Cells were stimulated with glutamate and glycine (3 μM each) in the presence of compounds at a range of concentrations. Curves for inhibition of the Ca 2+ response in GluN2A-expressing cells were derived from fits to the Hill equation using GraphPad Prism (v6.00 for Mac, GraphPad Software, La Jolla California USA). Whereas MPX-004 and MPX-007 achieve full inhibition of the GluN2A Ca 2+ response by ~ 3 μM, TCN-201 never inhibits more than ~40% of the response. Each data point is a mean (± standard deviation) of data from 20–86 experiments).

Article Snippet: The human GRIN1 gene transcript variant NR1-3 (Origene, SKU#: SC115601, Rockville, MD), GRIN2A gene (Origene, SKU#: SC326381), GRIN2B gene (B’SYS, Witterswil, Switzerland), GRIN2C gene (NCBI locus NM_000835, optimized for oocyte expression and synthesized by GenScript, Piscataway, NJ) and GRIN2D gene (NCBI locus NM_000836, sequence optimized and synthesized by GenScript) were linearized and transcribed using the mMessage mMachine kit (Life Technologies) and promoters SP6 (for GluN1) and T7 (for GluN2A-2D). cRNAs were mixed together at a ratio of 1 GluN1:2 GluN2.

Techniques: Inhibition, Expressing, Derivative Assay, Software, Standard Deviation

Journal: PLoS ONE

Article Title: MPX-004 and MPX-007: New Pharmacological Tools to Study the Physiology of NMDA Receptors Containing the GluN2A Subunit

doi: 10.1371/journal.pone.0148129

Figure Lengend Snippet: C ells were stimulated with glutamate and glycine (3 μM each) in the presence of compounds at a range of concentrations. IC 50 for inhibition of the Ca 2+ response in GluN2A-expressing cells was fitted to the Hill equation using CDD Vault. For GluN2B or GluN2D no IC 50 could be determined, so the effect of each compound at 10 μM is shown as the % inhibition of the Ca 2+ response (note that negative % inhibition represents an increase in Ca 2+ response over glutamate plus glycine alone). Values are the mean IC 50 or % ± SEM with the number of replicate curves indicated in parentheses. ND- not determined.

Article Snippet: The human GRIN1 gene transcript variant NR1-3 (Origene, SKU#: SC115601, Rockville, MD), GRIN2A gene (Origene, SKU#: SC326381), GRIN2B gene (B’SYS, Witterswil, Switzerland), GRIN2C gene (NCBI locus NM_000835, optimized for oocyte expression and synthesized by GenScript, Piscataway, NJ) and GRIN2D gene (NCBI locus NM_000836, sequence optimized and synthesized by GenScript) were linearized and transcribed using the mMessage mMachine kit (Life Technologies) and promoters SP6 (for GluN1) and T7 (for GluN2A-2D). cRNAs were mixed together at a ratio of 1 GluN1:2 GluN2.

Techniques: Inhibition, Expressing

Journal: PLoS ONE

Article Title: MPX-004 and MPX-007: New Pharmacological Tools to Study the Physiology of NMDA Receptors Containing the GluN2A Subunit

doi: 10.1371/journal.pone.0148129

Figure Lengend Snippet: Oocytes were exposed to MPX-004 or MPX-007 at concentrations from 10 nM to 10 μM as indicated. Inhibition curves were generated using GraphPad Prism and IC 50 values were generated using CCD Vault. The IC 50 s for inhibition of GluN2A-mediated currents were 198 ± 17 and 143 ± 10 nM for MPX-004 and MPX-007, respectively. Each data point is a mean (± standard deviation) of data from 4–12 oocytes.

Article Snippet: The human GRIN1 gene transcript variant NR1-3 (Origene, SKU#: SC115601, Rockville, MD), GRIN2A gene (Origene, SKU#: SC326381), GRIN2B gene (B’SYS, Witterswil, Switzerland), GRIN2C gene (NCBI locus NM_000835, optimized for oocyte expression and synthesized by GenScript, Piscataway, NJ) and GRIN2D gene (NCBI locus NM_000836, sequence optimized and synthesized by GenScript) were linearized and transcribed using the mMessage mMachine kit (Life Technologies) and promoters SP6 (for GluN1) and T7 (for GluN2A-2D). cRNAs were mixed together at a ratio of 1 GluN1:2 GluN2.

Techniques: Inhibition, Generated, Standard Deviation

Journal: PLoS ONE

Article Title: MPX-004 and MPX-007: New Pharmacological Tools to Study the Physiology of NMDA Receptors Containing the GluN2A Subunit

doi: 10.1371/journal.pone.0148129

Figure Lengend Snippet: Effect of MPX-004 on the ratio of NMDA receptor- to AMPA receptor-mediated synaptic currents (NMDAR/AMPAR ratio) at layer 4-to-2/3 synapses in mouse visual cortex.

Article Snippet: The human GRIN1 gene transcript variant NR1-3 (Origene, SKU#: SC115601, Rockville, MD), GRIN2A gene (Origene, SKU#: SC326381), GRIN2B gene (B’SYS, Witterswil, Switzerland), GRIN2C gene (NCBI locus NM_000835, optimized for oocyte expression and synthesized by GenScript, Piscataway, NJ) and GRIN2D gene (NCBI locus NM_000836, sequence optimized and synthesized by GenScript) were linearized and transcribed using the mMessage mMachine kit (Life Technologies) and promoters SP6 (for GluN1) and T7 (for GluN2A-2D). cRNAs were mixed together at a ratio of 1 GluN1:2 GluN2.

Techniques:

Journal: eLife

Article Title: Ring finger protein 10 is a novel synaptonuclear messenger encoding activation of NMDA receptors in hippocampus

doi: 10.7554/eLife.12430

Figure Lengend Snippet: ( A , B ) Mixed primary hippocampal cultures ( DIV14 ) immunolabeled with antibodies for RNF10 (red), glial marker GFAP (A; green) or the neuronal marker MAP2 (B; green), and Dapi (blue) to stain the nucleus; scale bar: 20 μm. ( C ) Dendrite of hippocampal neuron transfected with GFP-RNF10 ( DIV7 ) and immunolabeled at DIV14 for GFP (green), GluN2A (red) and PSD-95 (blue); scale bar: 3 μm. ( D ) High-magnification confocal images of neuronal dendrites ( DIV14 ) immunolabeled for endogenous RNF10 (green) and GluN2A (red; left panels) or PSD-95 (red; right panels); scale bar: 4 μm. ( E ) RNF10 and markers of the presynaptic (synaptophysin) and postsynaptic compartment (PSD-95, GluN2A) were analyzed by WB in various subcellular compartments (H: Homogenate fraction; S1: supernatant 1; P1: nuclear fraction; S2: cytosolic fraction 2; P2: crude membrane fraction 2; Syn: synaptosomal fraction; PSD1: Triton Insoluble postsynaptic fraction; PSD2: postsynaptic density fraction). ( F ) Representative confocal images of adult rat hippocampal CA1 pyramidal layer sections showing immunohistochemical labeling for RNF10 (red), MAP2 (green), and Dapi (blue); scale bar: 40 μm. ( G ) Confocal images of dendrites from hippocampal neurons ( DIV14 ) transfected at DIV7 with shGluN2A or scramble vector and immunolabeled for GluN2A; scale bar: 4 μm. The histogram shows the quantification of GluN2A integrated density in dendrites (n=7, **p=0.0069 scramble vs shGluN2A; unpaired Student’s t-test). ( H ) GluN2A silencing induces a reduction of RNF10 enrichment at the glutamatergic synapse. Confocal images of primary hippocampal neurons transfected with pGFP-V-RS-scramble (left panels) or with pGFP-V-RS-shGluN2A (right panels) plasmids and immunolabeled ( DIV14 ) for RNF10 (green) and PSD-95 (red); scale bar: 4 μm. The histogram shows the quantification of RNF10 co-localization with PSD-95-positive puncta (n=30, ***p<0.001; unpaired Student’s t-test). ( I ) Confocal images of dendrites from hippocampal neurons ( DIV14 ) transfected at DIV7 with shGluN2A or scramble vector and immunolabeled for surface GluN2A (blue) and RNF10 (red); scale bar: 4 μm. DOI: http://dx.doi.org/10.7554/eLife.12430.003

Article Snippet: To knock down rat GluN2A gene expression, a set of 4 short hairpin RNA (shRNA) expression vectors in pGFP-V-RS was purchased from Origene (Rockville, MD).

Techniques: Immunolabeling, Marker, Staining, Transfection, Immunohistochemical staining, Labeling, Plasmid Preparation

Journal: eLife

Article Title: Ring finger protein 10 is a novel synaptonuclear messenger encoding activation of NMDA receptors in hippocampus

doi: 10.7554/eLife.12430

Figure Lengend Snippet: ( A ) Co-immunoprecipitation (co-i.p.) assay performed in P2 crude membrane fractions by using antibodies against PSD-95, RNF10, synaptophysin (Syn) and GluN2A. WB analysis shows the levels of GluN2A (left panel) and GluN2B (right panel) in the co-immunoprecipitated material. No ab lane: control lane in absence of antibodies during the co-i.p. assay. ( B ) Jacob is a part of the GluN2B receptor complex. Affinity purified pan-Jacob antibodies co-immunoprecipitate GluN2B. ( C ) Co-i.p. assay performed by using an anti-RNF10 antibody from COS-7 cell extracts transfected with HA-GluN1 and GFP-GluN2A or GFP-GluN2B. WB analysis was performed by using anti-GFP and anti-RNF10 antibodies. No ab lane: control lane in absence of antibodies during the co-i.p. assay. ( D ) COS-7 cells expressing RNF10 were transfected with HA-GluN1 and GFP-GluN2A or GFP-GluN2B constructs and immunolabeled for GFP (green), GluN1 (blue), Dapi (cyan) and endogenous RNF10 (red); scale bar: 10 μm. ( E ) In situ detection of proximity between RNF10 and GluN2A (red) along MAP2 (green; left panels) or GFP-positive (green; right panels) dendrites. In control experiments (-), primary hippocampal neurons were labeled with only RNF10 primary antibody and thus only unspecific PLA signals are generated; scale bars: 5 μm (MAP2) and 3 μm (GFP). ( F , G ) COS-7 cells expressing RNF10 were transfected with GFP-GluN2A (right panels) or GFP-GluN2B (left panels) constructs and immunolabeled for GFP (green), Dapi (cyan) and endogenous RNF10 (red); scale bar: 10 μm. The histogram shows the quantification of RNF10 integrated density (i.d.) expressed as cytoplasm/nucleus ratio (n=10; ***p<0.001; one-way ANOVA, followed by Bonferroni post-hoc test). DOI: http://dx.doi.org/10.7554/eLife.12430.004

Article Snippet: To knock down rat GluN2A gene expression, a set of 4 short hairpin RNA (shRNA) expression vectors in pGFP-V-RS was purchased from Origene (Rockville, MD).

Techniques: Immunoprecipitation, Affinity Purification, Transfection, Expressing, Construct, Immunolabeling, In Situ, Labeling, Generated

Journal: eLife

Article Title: Ring finger protein 10 is a novel synaptonuclear messenger encoding activation of NMDA receptors in hippocampus

doi: 10.7554/eLife.12430

Figure Lengend Snippet: ( A ) Scheme showing GST-GluN2A fusion proteins used in the pull-down assay. ( B ) GST and GST-GluN2A fusion proteins were incubated in a pull-down assay with rat hippocampal extracts. WB analysis was performed with RNF10 antibody. ( C ) Confocal images of COS-7 cells transfected with GFP-GluN2A (1–1049) and immunolabeled for GFP (green), Dapi (cyan) and RNF10 (red); scale bar: 10 μm. ( D ) Co-i.p. assay performed from lysates of COS-7 cells transfected with GFP-GluN2A or GFP-GluN2A(1–1049). WB analysis was performed by using a GFP antibody (JL-8). ( E ) GST and GST-RNF10 full-length (FL) fusion proteins were incubated in a pull-down assay with rat hippocampal extracts in presence or absence of calcium (2 mM). WB analysis was performed with GluN2A antibody. ( F ) GST and GST-GluN2A(839–1464) fusion proteins were incubated in a pull-down assay with rat hippocampal extracts in presence or absence of calcium (2 mM). WB analysis was performed with RNF10 and CaM antibodies. ( G ) GST and GST-GluN2A(839–1049) fusion proteins were incubated in a pull-down assay with rat hippocampal extracts with CaM (0.1 μM) in the presence or absence of calcium (2 mM). WB analysis was performed with RNF10 and CaM antibodies. ( H ) Co-i.p. assay performed from lysates of COS-7 cells transfected with GFP, GFP-GluN2A or GFP-GluN2B in the presence or absence of calcium (2 mM)/CaM (0.1 μM). WB analysis was performed by using RNF10 and CaM antibodies. ( I ) Co-i.p assay performed by using a GFP antibody from lysates of COS-7 cells transfected with RNF10 FL, RNF10(221–802) and GFP-GluN2A. WB analysis was performed by using a Myc antibody. No ab lanes: control lanes in absence of antibodies during the co-i.p. assay. ( J ) Co-i.p assay performed by using a Myc antibody from lysates of COS-7 cells transfected with RNF10 truncation mutants and GFP-GluN2A. WB analysis was performed by using a GFP antibody (JL-8). ( K , L ) COS-7 cells expressing RNF10 were transfected with GFP-GluN2A and Myc-RNF10 FL or Myc-RNF10 (221–802) constructs and immunolabeled for GFP (green), Myc (red) and Dapi (blue) ( G ); scale bar: 10 μm. The histogram ( H ) shows the quantification of Myc/EGFP co-localization index [n=10; ***p<0.001 RNF10 FL vs RNF10(221–802); unpaired Student’s t-test]. ( M ) GST and GST-RNF10 fusion proteins were incubated in a pull-down assay with rat hippocampal extracts. WB analysis was performed with GluN2A antibody. DOI: http://dx.doi.org/10.7554/eLife.12430.005

Article Snippet: To knock down rat GluN2A gene expression, a set of 4 short hairpin RNA (shRNA) expression vectors in pGFP-V-RS was purchased from Origene (Rockville, MD).

Techniques: Pull Down Assay, Incubation, Transfection, Immunolabeling, Expressing, Construct

Journal: eLife

Article Title: Ring finger protein 10 is a novel synaptonuclear messenger encoding activation of NMDA receptors in hippocampus

doi: 10.7554/eLife.12430

Figure Lengend Snippet: ( A ) Confocal images of primary hippocampal neurons ( DIV14 ) transfected at DIV7 with pGIPZ-scramble, shRNF10 and shRNF10 plus flagRNF10 and immunolabeled for GFP (green); scale bar: 5 μm. ( B - E ) Histograms showing the quantification of dendritic spine density ( B ) (n=6–10; *p<0.05, scramble vs shRNF10; ***p<0.001, shRNF10 vs shRNF10 + flagRNF10; one-way ANOVA, followed by Tukey post-hoc test), dendritic spine length ( C ), dendritic spine head width ( D ) and dendritic spine type ( E ). ( F ) WB analysis from homogenates of primary hippocampal neurons ( DIV14 ) and organotypic hippocampal slices ( DIV14 ) lentivirally infected with pGIPZ-scramble sequence (scramble) as control or with pLKO-shRNF10 (shRNF10). The histogram shows the quantification of the expression levels of GluN2A, GluA1, PSD-95 and GluN2B in shRNF10-infected neurons and slices, normalized on tubulin and expressed as % of scramble (n=6; *p<0.05; **p<0.01; unpaired Student’s t-test). ( G ) mRNA expression levels of genes associated with synaptic transmission or dendritic spine morphology by real-time PCR from DIV14 organotypic hippocampal slices lentivirally infected ( DIV4 ) with pGIPZ-scramble sequence (scramble) as control or with pLKO-shRNF10 (shRNF10) (n=4, ***p<0.001; **p<0.01; *p<0.05; unpaired Student’s t-test). ( H ) WB for ArhGef6, ArhGap4, Ophn1 and tubulin from cell lysates of organotypic hippocampal slices infected with pGIPZ-scramble or with pLKO-shRNF10. The histogram shows the quantification of protein levels from shRNF10 samples with respect to pGIPZ-scramble, following normalization on tubulin (n=3, *p<0.05; **p<0.01; unpaired Student’s t-test). DOI: http://dx.doi.org/10.7554/eLife.12430.006

Article Snippet: To knock down rat GluN2A gene expression, a set of 4 short hairpin RNA (shRNA) expression vectors in pGFP-V-RS was purchased from Origene (Rockville, MD).

Techniques: Transfection, Immunolabeling, Infection, Sequencing, Expressing, Transmission Assay, Real-time Polymerase Chain Reaction

Journal: eLife

Article Title: Ring finger protein 10 is a novel synaptonuclear messenger encoding activation of NMDA receptors in hippocampus

doi: 10.7554/eLife.12430

Figure Lengend Snippet: ( A ) Hippocampal neurons ( DIV14 ) were incubated for 24 hr with 50 μM Bicuculline and 2.5 mM 4-AP ('Bic') and immunolabeled for RNF10 (green) and PSD-95 (red). The histogram shows the quantification of RNF10 levels along dendrites 24 hr after treatment (n=30, ***p<0.001; unpaired Student’s t-test); scale bar: 4 μm. ( B , C ) Bic treatment induces RNF10-tdEOS translocation from distal dendrites to the nucleus in hippocampal neurons. ( B ) The histogram shows a significant increase in RNF10-tdEOS photoconverted fluorescent intensities in the nucleus following Bic treatment (n=6; p<0.05 Bic vs. control, from 40' to 90'; unpaired Student’s t-test). ( C ), left panels: Baseline confocal image of RNF10-tdEOS expressing hippocampal neuron illuminated sequentially with 488 nm and 555 nm laser excitation wavelengths showing no emitted signal in the red spectra (ex555nm; left panels). Distal dendrite (ROI) selected for photoconversion was illuminated with UV laser (405 nm wavelengths) repetitively through the image z-stack. ( C ), right panels: Depicted are confocal max intensity projection images at respective time points in control after Bic treatment or in control (untreated) neurons; scale bar: 20 μm. ( D ) Hippocampal neurons ( DIV14 ) were treated with Bic in presence of the GluN2A inhibitor NVP-AAM007 at different concentrations (50 and 300 nM), immunolabeled for RNF10 (green) and stained with Dapi (blue). The histogram shows the quantification of RNF10 integrated density in the nucleus expressed as % of control neurons (n=10, ***p<0.001 control vs Bic, Bic vs Bic+NVP 50 nM and Bic vs Bic+NVP 300 nM; one-way ANOVA followed by Bonferroni post-hoc test); scale bar: 10 μm. ( E ) Hippocampal neurons ( DIV14 ) treated with 'Syn' (50 μM Bicuculline; 2.5 mM 4-AP; 5 μM Ifenprodil, 8 hr), with 'Extrasyn#1' or 'Extrasyn#2' protocols (see Materials and methods), immunolabeled for RNF10 (green) and stained with Dapi (blue). Histogram showing the quantification of RNF10 integrated density in the nucleus expressed as % of control neurons (n=8, *p<0.05 Extrasyn#2 vs Syn, **p<0.01 Syn vs Extrasyn#1 and Syn vs. control; one-way ANOVA followed by Bonferroni post-hoc test); scale bar: 10 μm. ( F ) Depicted are representative laserscans averaged from three confocal sections of the nucleus of DIV16 hippocampal primary neurons immunolabeled with affinity purified antibodies against pan-Jacob (rabbit) and co-labeled with anti-MAP2 antibodies as a neuronal specific marker. Neuronal nuclei are outlined with the DNA stain Hoechst 34580. Scale bar: 10 µm. Relative fluorescence intensities of Jacob 30 min of synaptic stimulation with and without selective inhibitors were normalized to untreated non-stimulated control (n=31–70, ***p<0.001; one-way ANOVA followed by Bonferroni post-hoc test). DOI: http://dx.doi.org/10.7554/eLife.12430.008

Article Snippet: To knock down rat GluN2A gene expression, a set of 4 short hairpin RNA (shRNA) expression vectors in pGFP-V-RS was purchased from Origene (Rockville, MD).

Techniques: Incubation, Immunolabeling, Translocation Assay, Expressing, Staining, Affinity Purification, Labeling, Marker, Fluorescence

Journal: eLife

Article Title: Ring finger protein 10 is a novel synaptonuclear messenger encoding activation of NMDA receptors in hippocampus

doi: 10.7554/eLife.12430

Figure Lengend Snippet: ( A ) Confocal images of primary hippocampal neurons transfected with Myc-RNF10 full-length (FL) or Myc-RNF10 truncated mutants and immunolabeled for Myc (green) and Dapi (blue); scale bar: 20 μm. ( B ) Primary hippocampal neurons transfected with Myc, Myc-RNF10 (1–611), pGIPZ-scramble or with shRNF10 were immunolabeled ( DIV14 ) for PSD-95. The histograms show the quantification of PSD-95-positive puncta expressed as % of Myc (upper histogram) or scramble (lower histogram) transfected neurons (n=50–88, ***p<0.001, Myc-RNF10 (1–611) vs Myc and shRNF10 vs scramble; unpaired Student’s t-test); scale bar: 4 μm. ( C ) Confocal images of dendrites of hippocampal neurons ( DIV14 ) immunolabeled for RNF10 (red), surface GluN2A (green) and importin α1 (blue); scale bar: 4 μm. ( D ) Importin α1 and PSD-95 protein levels were analyzed by means of WB analysis in various subcellular compartments purified from rat hippocampal tissue (H: Homogenate fraction, S1: supernatant 1, P1: nuclear fraction, S2: cytosolic fraction 2, P2: crude membrane fraction 2, Syn: synaptosomal fraction, PSD1: Triton insoluble postsynaptic fraction, PSD2: postsynaptic density fraction). ( E ) Representative co-i.p. assay showing the interaction between RNF10 and importin α1 in hippocampal tissue homogenate (upper and lower panel) and PSD fraction (lower panel). No Ab lane: control lane in absence of the antibody. WB analysis was performed with RNF10 and importin α1 antibodies. ( F ) RNF10-tagged with GFP was co-expressed with multiple importin α isoforms ( KPNA1-KPNA6 ) tagged with tagRFP in HEK293T cells. RNF10-GFP was immunoprecipitated from cell extract using anti-GFP MicroBeads. Co-immunoprecipitated importinerase - α isoforms were detected in complex with RNF10 using anti-tagRFP antibodies. ( G ) Co-i.p. assay performed by using an importin α1 antibody from cell homogenates of control (C) neurons or treated with Bic. WB analysis was performed with RNF10 antibody. The histogram shows the quantification of RNF10/importin α1 interaction expressed as % of control (n=3, *p<0.05; unpaired Student’s t-test). ( H ) Representative co-i.p. assay from cell homogenates of control (C) neurons or treated with Bic. WB analysis was performed with RNF10 antibody. The histogram shows the quantification of RNF10 interaction with GluN2A and Meox2 expressed as % of control (n=3, ***p<0.001, Meox2, Bic vs control; **p<0.01, GluN2A, Bic vs control; unpaired Student’s t-test). DOI: http://dx.doi.org/10.7554/eLife.12430.009

Article Snippet: To knock down rat GluN2A gene expression, a set of 4 short hairpin RNA (shRNA) expression vectors in pGFP-V-RS was purchased from Origene (Rockville, MD).

Techniques: Transfection, Immunolabeling, Purification, Immunoprecipitation

Journal: eLife

Article Title: Ring finger protein 10 is a novel synaptonuclear messenger encoding activation of NMDA receptors in hippocampus

doi: 10.7554/eLife.12430

Figure Lengend Snippet: ( A–C ) RNF10-tdEOS translocates from distal dendrites to the nucleus in mature hippocampal neurons upon high-frequency 18s@50Hz field synaptic stimulation. Distal dendrites (ROIs) selected for photoconversion were illuminated with UV laser (405 nm wavelengths) repetitively through the image z-stack (image t0`). ( A ) Representative images of control and stimulated RNF10-tdEOS expressing hippocampal neurons illuminated with 568 nm laser excitation wavelength 1 min following photoconversion. ( B ) The histogram shows a significant increase in RNF10-tdEOS photoconverted fluorescent intensities in the nucleus following high frequency 18s@50Hz field synaptic stimulation (n=9, *p<0.05, **p<0.01, ***p<0.001; unpaired Student’s t-test). ( C ) Depicted are confocal max intensity projection images of control and stimulated RNF10-tdEOS expressing hippocampal neurons at respective time points after stimulation. Lower panels: magnified image sequence of UV illuminated dendritic segment with multiple spines is represented showing the decrease in distal dendrites upon field stimulation. All experiments were performed in a presence of anisomycin (7.5 µM). ( D ) Confocal images of dendrites of hippocampal neurons ( DIV14 ) transfected with GFP (green) to visualize neurites and immunolabeled for RNF10 (red) and PSD-95 (blue). The histogram shows the quantification of RNF10 signal in dendrites 2 hr after induction of cLTP (see Materials and methods) expressed as % of control (n=5, **p<0.01; unpaired Student’s t-test); scale bar: 4 μm. ( E ) Co-i.p. assay from hippocampal extracts performed with an antibody against GluN2A. WB analysis was performed using antibody for RNF10 and GluN2A. The graph shows the effect of cLTP induction on RNF10 interaction with GluN2A expressed as % of control (n=4, ***p<0.001; unpaired Student’s t-test). ( F ) Representative confocal images of hippocampal neurons ( DIV14 ) after the induction of cLTP in the presence or absence of 7.5 μM anisomycin and immunolabeled for RNF10 (green) and Dapi (blue); scale bar: 10 μm. ( G ) WB analysis for RNF10 from P1 nuclear fraction purified from hippocampal neurons after the induction of cLTP in the presence or absence of 7.5 μM anisomycin. The histogram shows the quantification of RNF10 integrated density normalized on Histone-H3 (n=4, *p<0.05, control vs cLTP and control vs cLTP+Anisomycin; one-way ANOVA, followed by Tukey post-hoc test). DOI: http://dx.doi.org/10.7554/eLife.12430.010

Article Snippet: To knock down rat GluN2A gene expression, a set of 4 short hairpin RNA (shRNA) expression vectors in pGFP-V-RS was purchased from Origene (Rockville, MD).

Techniques: Expressing, Sequencing, Transfection, Immunolabeling, Purification

Journal: eLife

Article Title: Ring finger protein 10 is a novel synaptonuclear messenger encoding activation of NMDA receptors in hippocampus

doi: 10.7554/eLife.12430

Figure Lengend Snippet: ( A ) Confocal images from living hippocampal neurons transfected with dTomato ( DIV10 ) and treated with TAT-TF2 or NLS2-TF2 coniugated peptide ( DIV14 ). Samples were illuminated with 543 nm and 488 nm to visualize respectively neurons and peptide. The representative image shows the presence of peptide (green) within the neuron (red, DTOMATO), demonstrating the capability of crossing the plasmatic membrane; scale bar: 10 μm. ( B ) Representative co-i.p. assay performed by using anti-RNF10 antibody and showing the interaction between RNF10 and importin α1 in primary hippocampal neurons ( DIV14 ) treated with NLS2 peptide (active) or TAT (control) peptide. No IgG lane: control lane in absence of the antibody. WB analysis was performed with importin α1 and RNF10 antibodies. The histogram shows the quantification of importin α1 interaction with RNF10 expressed as % of control (TAT; n=3; *p<0.05; unpaired Student’s t-test). ( C , E ) Hippocampal neurons ( DIV14 ) were treated with NLS2 peptide (active) or TAT (inactive) peptide for 24 hr and then cLTP was induced in the presence of the same peptides. Confocal images show the immunolabeling for RNF10 (green) and the staining for Dapi (blue) in the nucleus ( C ) or PSD-95 (red) along dendrites ( E ); scale bars: 10 μm ( C ) and 4 μm ( E ). ( D , F ) The histograms show the quantification of RNF10 signal in the nucleus ( D ) and along dendrites ( F ) after the induction of cLTP in the presence of TAT or NLS2 peptides expressed as % of control [n=10,11 (D), n=17–19 (F)]; *p<0.05; **p<0.01; *p<0.001; one-way ANOVA, followed by Tukey post-hoc test). ( G , H ) WB analysis for RNF10 from P1 crude nuclear fraction purified from hippocampal neurons after the induction of cLTP in the presence of NLS2 or TAT peptide ( G ). The histogram ( H ) shows the quantification of RNF10 integrated density normalized on Histone-H3 and expressed as % of control (n=4, *p<0.05; one-way ANOVA, followed by Tukey post-hoc test). ( I ) Representative co-i.p. assay performed with an antibody against RNF10 from cell lysates of hippocampal neurons following induction of cLTD. WB analysis was performed using antibody for RNF10 and GluN2A. ( J ) Confocal images of the soma of hippocampal neurons ( DIV14 ) treated with a protocol to induce cLTD and then immunolabeled for RNF10 (green) and stained with Dapi (blue). Scale bar: 10 μm. DOI: http://dx.doi.org/10.7554/eLife.12430.011

Article Snippet: To knock down rat GluN2A gene expression, a set of 4 short hairpin RNA (shRNA) expression vectors in pGFP-V-RS was purchased from Origene (Rockville, MD).

Techniques: Transfection, Immunolabeling, Staining, Purification

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: bioRxiv

Article Title: Dose-Dependent Induction Of CPP Or CPA By Intra-pVTA Ethanol: Role Of Mu Opioid Receptors And Effects On NMDA Receptors

doi: 10.1101/2020.01.07.897702

Figure Lengend Snippet: Ethanol place preferences is associated with the expression of mRNA from GluN2A but not from GluN1 subunit in the NAc. A , Schematic of the experimental design. B , Place preference elicited by the administration of 70 nmol of ethanol in the pVTA. Data are mean ± SEM represented as preference score (test minus pretest time spent in ethanol-paired compartment). * denotes significant differences between means (p<0.01, t-Test). C , Diagram of brain coronal sections indicating in blue the area of all microinjections in pVTA. D , Expression of mRNA from NMDA subunits GluN1 and GluN2A in NAc after ethanol induced CPP. Data are mean ± SEM represented as number of dots (mRNA molecules) per cell. * denotes significant differences between means (p<0.05). E , Expression of mRNA from NMDA subunits GluN1 and GluN2A in hippocampus after ethanol induced CPP. Data are mean ± SEM represented as number of dots (mRNA molecules) per cell.

Article Snippet: Membranes were then blocked in 5% non-fat dried milk in TBS-Tween-20 (TBS-T) 0.1% (20mM Tris and 500mM NaCl pH 7.5) and incubated overnight at 4°C with the primary antibody: anti-GluN1 (1:1000; Merck KGaA, Darmstadt, Germany) and anti-GluN2A monoclonal antibody (1:1000; Merck KGaA, Darmstadt, Germany).

Techniques: Expressing

Journal: bioRxiv

Article Title: Dose-Dependent Induction Of CPP Or CPA By Intra-pVTA Ethanol: Role Of Mu Opioid Receptors And Effects On NMDA Receptors

doi: 10.1101/2020.01.07.897702

Figure Lengend Snippet: Representative pictures of the compilation files obtained in the mRNA In Situ Hybridization Assay used for the quantification of mRNA from GluN1 and GluN2A subunits in NAc (left) and hippocampus (right) after ethanol induced CPP. Squares delimit the area of the amplified pictures shown in for the NAc and in for the hippocampus. Abbreviatures: Aca , anterior commissure; CA3 , field CA3 of hippocampus; DG , dentate gyrus; CA1 , field CA1 of hippocampus.

Article Snippet: Membranes were then blocked in 5% non-fat dried milk in TBS-Tween-20 (TBS-T) 0.1% (20mM Tris and 500mM NaCl pH 7.5) and incubated overnight at 4°C with the primary antibody: anti-GluN1 (1:1000; Merck KGaA, Darmstadt, Germany) and anti-GluN2A monoclonal antibody (1:1000; Merck KGaA, Darmstadt, Germany).

Techniques: In Situ Hybridization, Amplification

Journal: bioRxiv

Article Title: Dose-Dependent Induction Of CPP Or CPA By Intra-pVTA Ethanol: Role Of Mu Opioid Receptors And Effects On NMDA Receptors

doi: 10.1101/2020.01.07.897702

Figure Lengend Snippet: Representative amplified pictures obtained in the mRNA In Situ Hybridization Assay used for the quantification of mRNA from GluN1 and GluN2A subunits in NAc. Amplified pictures from one animal of each group (aCSF on the left and ethanol 70 nmol on the right) were selected.

Article Snippet: Membranes were then blocked in 5% non-fat dried milk in TBS-Tween-20 (TBS-T) 0.1% (20mM Tris and 500mM NaCl pH 7.5) and incubated overnight at 4°C with the primary antibody: anti-GluN1 (1:1000; Merck KGaA, Darmstadt, Germany) and anti-GluN2A monoclonal antibody (1:1000; Merck KGaA, Darmstadt, Germany).

Techniques: Amplification, In Situ Hybridization

Journal: bioRxiv

Article Title: Dose-Dependent Induction Of CPP Or CPA By Intra-pVTA Ethanol: Role Of Mu Opioid Receptors And Effects On NMDA Receptors

doi: 10.1101/2020.01.07.897702

Figure Lengend Snippet: Representative amplified pictures obtained in the mRNA In Situ Hybridization Assay used for the quantification of mRNA from GluN1 and GluN2A subunits in hippocampus. Amplified pictures from one animal of each group (aCSF on the left and ethanol 70 nmol on the right) were selected.

Article Snippet: Membranes were then blocked in 5% non-fat dried milk in TBS-Tween-20 (TBS-T) 0.1% (20mM Tris and 500mM NaCl pH 7.5) and incubated overnight at 4°C with the primary antibody: anti-GluN1 (1:1000; Merck KGaA, Darmstadt, Germany) and anti-GluN2A monoclonal antibody (1:1000; Merck KGaA, Darmstadt, Germany).

Techniques: Amplification, In Situ Hybridization

Journal: bioRxiv

Article Title: Dose-Dependent Induction Of CPP Or CPA By Intra-pVTA Ethanol: Role Of Mu Opioid Receptors And Effects On NMDA Receptors

doi: 10.1101/2020.01.07.897702

Figure Lengend Snippet: Simplified representation of the dose-dependent ethanol effect on place preference or place aversion and its hypothesized mechanism. The administration of low doses of ethanol into the VTA does not induce preference or aversion for the drug paired compartment. Medium doses elicit CPP, presumably through an increase of the ethanol metabolized fraction. In our hypothesis and based in previous literature, the ethanol metabolite, salsolinol, would activate MORs, decreasing GABA release and finally increasing DA release in the NAc by a disinhibition of VTA DA neurons. The expression of ethanol induced CPP also results in an increase of mRNA form GluN2A in the NAc. Moreover, the blockade of the MORs in the VTA inhibits the acquisition of this ethanol induced CPP. However, the administration of high doses results in the development of CPA. In this case, the effect of the non-metabolized fraction would predominate and the inhibition of the VTA DA neurons would result in a decrease of DA release in the NAc. In orange GABA terminals and in purple VTA DA neurons. MOR: mu opioid receptor; GABAR: gamma-amino butyric acid (GABA) receptor; NAc: nucleus accumbens; VTA: ventral tegmental area; DA: dopamine; GLUN2A: NMDA receptor subunit 2A.

Article Snippet: Membranes were then blocked in 5% non-fat dried milk in TBS-Tween-20 (TBS-T) 0.1% (20mM Tris and 500mM NaCl pH 7.5) and incubated overnight at 4°C with the primary antibody: anti-GluN1 (1:1000; Merck KGaA, Darmstadt, Germany) and anti-GluN2A monoclonal antibody (1:1000; Merck KGaA, Darmstadt, Germany).

Techniques: Expressing, Inhibition