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

Journal: eLife

Article Title: Co-agonists differentially tune GluN2B-NMDA receptor trafficking at hippocampal synapses

doi: 10.7554/eLife.25492

Figure Lengend Snippet: ( A ) Diagram representing the Quantum Dot (QD) coupled to the specific antibody used to track single surface GluN2-NMDAR (top). Example of a synapse area (Syn) identified after incubation with MitoTracker (bottom). Scale: 1 µm. ( B ) Examples of the surface trajectories of single QD-coupled GluN2A-NMDAR in the synaptic area. Scale: 150 nm. ( C ) Mean diffusion coefficient of synaptic GluN2A-QD normalized to control condition (before co-agonist or enzyme application). Glycine: n = 8,115, D-serine: n = 5,555, Bs GO: n = 308, Rg DAAO: n = 121 trajectories; p<0.0001 Kruskal-Wallis test. ( D ) Mean Square Displacement (MSD) of surface GluN2A trajectories measured either on the presence of glycine or D-serine. ( E ) Examples of the surface trajectories of single QD-coupled GluN2B-NMDAR as in ( B ). ( F ) Mean diffusion coefficient of synaptic GluN2B-QD normalized to control condition. Glycine: n = 318, D-serine: n = 836, Bs GO: n = 73, Rg DAAO: n = 605 trajectories; p<0.0001 Kruskal-Wallis test. ( G ) MSD of surface GluN2B trajectories as in ( D ). ( H ) Transition ratio (i.e. entries or exits from synaptic areas/total number of trajectories per cellular field) of GluN2B-QD in the presence of either glycine or D-serine. Control: n = 7, glycine: n = 4, D-serine: n = 7 neuronal fields; p=0.1187 Kruskal-Wallis test. ( I–J ) Synaptic fraction of QD-detected GluN2A-NMDAR ( I ) and GluN2B-NMDAR ( J ) in the synaptic area, normalized to the respective controls. GluN2A: n = 21 glycine, n = 21 D-serine, n = 16 BsGO, n = 22 Rg DAAO dendritic fields; p=0.9974 Kruskal-Wallis test. GluN2B: n = 8 glycine, n = 14 D-serine, n = 37 BsGO, n = 15 Rg DAAO dendritic fields; p<0.0001 Kruskal-Wallis test. Data are represented as mean ± s.e.m.; ***p<0.0001, **p<0.001, *p<0.05, Dunn's Multiple Comparison Test. DOI: http://dx.doi.org/10.7554/eLife.25492.002

Article Snippet: After the treatment, NMDAR were surface stained by live-staining the cells with specific antibodies against the extracellular terminal of GluN2B or GluN2A (homemade antibodies 2 mg/ml, Agro-Bio, La Ferté Saint Aubin, France, 1:200), prepared in conditioned medium, 10 min at 37°C.

Techniques: Incubation, Diffusion-based Assay

Journal: eLife

Article Title: Co-agonists differentially tune GluN2B-NMDA receptor trafficking at hippocampal synapses

doi: 10.7554/eLife.25492

Figure Lengend Snippet: ( A ) Surface labeling of GluN2A in hippocampal neurons 18–19 div in culture stimulated with glycine or D-serine. Homer 1c staining was used as the synaptic maker. Scale: 10 µm. ( B ) Surface (extra- and synaptic) and synaptic GluN2A clusters overlapping Homer 1c positive clusters, normalized to the respective control conditions. Control: n = 41, glycine: n = 39, D-serine: n = 40 cells; surface GluN2A p=0.8935, synaptic GluN2A p=0.8830, Kruskal-Wallis test. ( C ) Total (intracellular and surface) GluN2A clusters from hippocampal neurons 17 div, labeled after permeabilization, stimulated with glycine or D-serine and normalized to the control. Control: n = 30, glycine n = 30, D-serine n = 30 cells; p=0.6884, Kruskal-Wallis test. ( D ) Surface labeling of GluN2B in hippocampal neurons 18–19 div in culture stimulated with glycine or D-serine. Homer 1c staining was used as the synaptic maker. Scale: 10 µm. ( E ) Surface (extra- and synaptic) and synaptic GluN2B clusters overlapping Homer 1c positive clusters, normalized to the respective control conditions. Control: n = 41, glycine: n = 39, D-serine: n = 40 cells; surface GluN2B p=0.1383, synaptic GluN2B p=0.0247, Kruskal-Wallis test followed by Dunn's Multiple Comparison Test, *p<0.05. ( F ) Total (intracellular and surface) GluN2B clusters from hippocampal neurons 17 div, labeled after permeabilization, stimulated with glycine or D-serine and normalized to the control. Control: n = 30, glycine n = 30, D-serine n = 30 cells; p=0.5478, Kruskal-Wallis test. ( G ) Synaptosomes were purified by subcellular fractionation from rats P30 hippocampi incubated for 60 min in aCSF containing either glycine or D-serine. ( H ) 1 µg of protein was probed against GluN2B, GluN2A and GluN1. Synaptophysin was used as a loading control. ( I ) Synaptic fraction of NMDAR subunits levels, calculated as the variation between the protein expression levels in glycine and in D-serine conditions, normalized to the non-treated condition, control. n = 5, GluN1 levels p=0.4409, GluN2A levels p=0.1062, GluN2B levels p=0.0207, Repeated measures ANOVA followed by Bonferroni's multiple comparison test, *p<0.05. Data are represented as mean ± s.e.m. DOI: http://dx.doi.org/10.7554/eLife.25492.003

Article Snippet: After the treatment, NMDAR were surface stained by live-staining the cells with specific antibodies against the extracellular terminal of GluN2B or GluN2A (homemade antibodies 2 mg/ml, Agro-Bio, La Ferté Saint Aubin, France, 1:200), prepared in conditioned medium, 10 min at 37°C.

Techniques: Labeling, Staining, Purification, Fractionation, Incubation, Expressing

Journal: eLife

Article Title: Co-agonists differentially tune GluN2B-NMDA receptor trafficking at hippocampal synapses

doi: 10.7554/eLife.25492

Figure Lengend Snippet: ( A ) Examples of the surface trajectories of single QD-coupled GluN2B-NMDAR in the synaptic area. Pre-incubated with scramble peptide (TAT-NS, top) or GluN2B PDZ-proteins disrupting binding (TAT-GluN2B, bottom), before (control, left) or after D-serine application (right). Scale: 150 nm. ( B ) Mean diffusion coefficient of synaptic GluN2B-QD normalized to respective control condition (before D-serine application). TAT-NS control: n = 543, TAT-NS + D-serine: n = 299, TAT-GluN2B control: n = 413, TAT-GluN2B + D-serine: n = 713 trajectories; TAT-NS p=0.0398, TAT-GluN2B p=0.9292, Mann-Whitney test. ( C ) MSD of surface GluN2B trajectories in the presence of either TAT-NS or TAT-GluN2B, with or without D-serine. ( D ) Surface labelling of anti-GluN2B in hippocampal neurons 16–17 div in culture after incubation with either TAT-NS (top) or TAT-GluN2B (bottom), with (right) or without D-serine (left). Homer 1c staining was used as the synaptic maker. Scale: 10 µm. ( E ) Mean intensity of synaptic surface GluN2B clusters (juxtapose to Homer1c clusters) normalized to the respective control. TAT-NS control: n = 41, TAT-NS + D-serine: n = 38, TAT-GluN2B control: n = 61, TAT-GluN2B + D-serine: n = 70 cells; TAT-NS p<0.0001, TAT-GluN2B p=0.3090, Mann-Whitney test. ( F–G ) GluN2B immunoprecipitates (GluN2B IP, ( F ) or GluN2A (GluN2A IP, ( G ) were probed against PSD-95 (top) or SAP-102 (bottom). PSD-95 and SAP-102 co-immunoprecipitation levels (co-IP) normalized to GluN2B ( F ) or GluN2A ( G ) levels after glycine or D-serine incubation. Glycine: n = 10, D-serine: n = 8; PSD-95 co-IP GluN2B p=0.0148; glycine and D-serine: n = 10; SAP-102 co-IP GluN2B p=0.9118; glycine and D-serine: n = 8; PSD-95 co-IP GluN2A p=0.8785, Mann-Whitney test. Data are represented as mean ± s.e.m.; *p<0.05, ***p<0.0001. DOI: http://dx.doi.org/10.7554/eLife.25492.005

Article Snippet: After the treatment, NMDAR were surface stained by live-staining the cells with specific antibodies against the extracellular terminal of GluN2B or GluN2A (homemade antibodies 2 mg/ml, Agro-Bio, La Ferté Saint Aubin, France, 1:200), prepared in conditioned medium, 10 min at 37°C.

Techniques: Incubation, Binding Assay, Diffusion-based Assay, MANN-WHITNEY, Staining, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: eLife

Article Title: Co-agonists differentially tune GluN2B-NMDA receptor trafficking at hippocampal synapses

doi: 10.7554/eLife.25492

Figure Lengend Snippet: ( A ) Schematic representation of experimental design (top). Hippocampal neurons 14 div, transfected at 10 div with: GluN1-GFP (donor) or GluN1-GFP plus GluN1-mCherry (acceptor, bottom). Both conditions were co-transfected with GluN2B-Flag. Scale: 10 µm. ( B ) Example of GluN1-GFP fluorescent (left) and FLIM image (right). NMDAR clusters (spines clusters: arrows; shaft clusters: arrow heads, top) lifetime in nanoseconds (ns) quantification (bottom). Scale: 10 µm, insert 1 µm. Schematic representation of lifetime decay of a donor-only (full line) and of the donor in the presence of the acceptor (dashed line, bottom left). Comparison between GluN1-GFP lifetime alone (donor-only), co-transfected with GluN1-mcherry with a ratio 1:3 or 1:2 (donor-acceptor pair) and co-transfected with GluN2B-mCherry (negative control). GluN1-GFP: n = 70, GluN1-GFP + GluN1-mCherry (1:3): n = 88, GluN1-GFP + GluN1-mCherry (1:2): n = 44, GluN1-GFP + GluN2B-mCherry: n = 60 spines and shaft clusters; p<0.0001, One-way analysis of variance, followed by Dunnett's Multiple Comparison Test, ***p<0.0001 (bottom right). ( c ) Example of FLIM image of GluN1-GFP/GluN1-mCherry clusters after addition of tyrode (control) or NMDA (left). Quantification of GluN1-GFP lifetime (right). Tyrode: n = 291, NMDA: n = 124; p=0.0165, F = 1.719, Unpaired t test, one-tail. ( D ) Example of FLIM image of GluN1-GFP/GluN1-mCherry clusters after addition of D-serine or glycine (top) in tyrode only or in the presence of NMDA (bottom). Quantification of GluN1-GFP lifetime change (lifetime after minus the lifetime before co-agonist addition, right). Tyrode: n = 291, glycine: n = 110, D-serine: n = 103, glycine in NMDA: n = 268, D-serine in NMDA: n = 233 spine and shaft clusters; tyrode p=0.4234, r 2 = 0.0001, glycine p=0.3650, r 2 = 0.0011, D-serine p=0.0255, r 2 = 0.0368, glycine in NMDA p=0.0949, r 2 = 0.0065, D-serine in NMDA p=0.4348, r 2 = 0.0001, Paired t-test, one-tail, before and after, *p<0.05. Data are represented as mean ± s.e.m. ( E ) Schematic representation of the c-terminus tails of the NMDAR in basal conditions (tyrode, ( i ) or in the presence of NMDA (ii), the co-agonists alone (iii, iv) or in activating conditions (co-agonists together with NMDAR, ( v, vi ). DOI: http://dx.doi.org/10.7554/eLife.25492.006

Article Snippet: After the treatment, NMDAR were surface stained by live-staining the cells with specific antibodies against the extracellular terminal of GluN2B or GluN2A (homemade antibodies 2 mg/ml, Agro-Bio, La Ferté Saint Aubin, France, 1:200), prepared in conditioned medium, 10 min at 37°C.

Techniques: Transfection, Negative Control

Journal: eLife

Article Title: Co-agonists differentially tune GluN2B-NMDA receptor trafficking at hippocampal synapses

doi: 10.7554/eLife.25492

Figure Lengend Snippet: ( A ) D-serine and glycine dose-response curves for GluN2A-NMDARs and GluN2B-NMDARs expressed in Xenopus oocytes. n and p values can be found in . ( B ) Average contents of glycine and D-serine, and D-serine/glycine ratio, measured by capillary electrophoresis in slices homogenates across development. n and p values can be found in . DOI: http://dx.doi.org/10.7554/eLife.25492.010

Article Snippet: After the treatment, NMDAR were surface stained by live-staining the cells with specific antibodies against the extracellular terminal of GluN2B or GluN2A (homemade antibodies 2 mg/ml, Agro-Bio, La Ferté Saint Aubin, France, 1:200), prepared in conditioned medium, 10 min at 37°C.

Techniques: Electrophoresis

Journal: eLife

Article Title: Co-agonists differentially tune GluN2B-NMDA receptor trafficking at hippocampal synapses

doi: 10.7554/eLife.25492

Figure Lengend Snippet: ( A ) In basal conditions GluN2A-NMDAR are enriched at synapses, whereas GluN2B-NMDAR are highly mobile. ( B ) This dynamics is maintained after glycine application. ( C ) However, D-serine application leads to a specific decrease of GluN2B-NMDAR surface diffusion and synaptic content, through the modulation of the receptors C-terminus interactions, possibly leading to an increase of the receptor internalization. DOI: http://dx.doi.org/10.7554/eLife.25492.011

Article Snippet: After the treatment, NMDAR were surface stained by live-staining the cells with specific antibodies against the extracellular terminal of GluN2B or GluN2A (homemade antibodies 2 mg/ml, Agro-Bio, La Ferté Saint Aubin, France, 1:200), prepared in conditioned medium, 10 min at 37°C.

Techniques: Diffusion-based Assay

Journal: bioRxiv

Article Title: Astrocyte GluN2C NMDA receptors control basal synaptic strengths of hippocampal CA1 pyramidal neurons in the stratum radiatum

doi: 10.1101/2021.05.28.446253

Figure Lengend Snippet: ( A ) Top: patch RT-PCR strategy. Bottom: representative voltage responses to current steps in CA1 pyramidal neuron (far left, black), and current responses to voltage steps in astrocytes in stratum radiatum (SR, green), stratum oriens (SO, yellow), and stratum lacunosum moleculare (SLM, blue). Scale bars: neuron, 40 mV, 500 ms; astrocytes, 6 nA, 200 ms. ( B ) Summary of I-V curves of astrocytes obtained before collecting RNA. N = 39 cells, 36 slices, 9 mice for each layer. ( C ) Summary of RT-PCR analysis for mRNA encoding indicated NMDAR subunits: GRIN1 (1), GRIN2A (2A), GRIN2B (2B), and GRIN2C (2C). Control samples (no patch) were obtained by inserting the electrode into the slice without patching cells. GRIN2D levels were undetectable in all cells examined (data not shown). ( D ) Western blots of co-immunoprecipitations performed using adult mouse hippocampal extracts with the indicated antibodies where anti-Cre antibody is used as a negative control. The blots are probed for GluN2A/2B (top row), GluN2C (middle row) or GluN1 (bottom row). ( E ) Representative brain sections of GRIN2C -Cre mice crossed to a tdTomato reporter line (Ai9), showing hippocampal area CA1 immunolabelled for GFAP, tdTomato and NeuN; scale bars, 160 μm (top), 25 μm (bottom). ( F ) Quantification of % cells that are positive for tdTomato amongst NeuN-labelled cells in stratum pyramidale (left) and SR (middle) and GFAP-labelled cells (right).

Article Snippet: Antibodies used were: mouse anti-NR1 (Synaptic Systems #114011), rabbit anti-GluN2A/2B (Synaptic Systems #244003), rabbit anti-GluN2C (generously provided by Dr. Masahiko Watanabe or purchased from Frontier Institute #GLURE3C-RB-AF270), mouse anti-Cre (Merck Millipore clone 2D8) and mouse anti-BirA (Novus biologicals #NBP2-59939).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Negative Control

Journal: Journal of Biomedical Science

Article Title: Generational synaptic functions of GABA A receptor β3 subunit deteriorations in an animal model of social deficit

doi: 10.1186/s12929-022-00835-w

Figure Lengend Snippet: Increased glutamatergic modifications across generations of the VPA-induced offspring. a–d Representative western blot profile and the protein levels of GluN2A ( a ), GluN2B ( b ), GluA1 ( c ), and GluA2 ( d ) in synaptoneurosomes from the amygdala. n = 5–6 rats from three or four litters for each condition. e , f Top, Representative EPSC traces at different stimuli. Scale bars, 100 pA, 50 ms. Bottom, input–output curves of NMDAR-EPSC ( e ) and AMPAR-EPSC ( f ) in response to a series of increasing stimulus intensities in the BLA pyramidal neurons. n = 6–7 cells from 4–5 rats from three or four litters for each condition. g Representative traces and bar graph show the AMPAR- to NMDAR-EPSC ratio measured from each group. Scale bars, 100 pA, 50 ms. n = 8–11 cells from 4–5 rats from three or four litters for each condition. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01 vs. indicated control; one-way ANOVA or two-way rmANOVA with Bonferroni post-hoc

Article Snippet: For GABA A R insertion, N -methyl- d -aspartate (NMDA) was purchased from Tocris (Minneapolis, MN), which was dissolved in artificial cerebrospinal fluid (aCSF) to obtain a concentration of 20 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\upmu }$$\end{document} μ M. For western blotting, the following antibodies of GluN2A (1:5000; Genetex; GTX63442), GluN2B (1:5000; Abcam; ab65783), GluA1 (1:5000; Abcam; ab109450), GluA2 (1:5000; Millipore; MAB397), gephyrin (1:5000; Alomone; AIP-005), GABA A R β3 subunit (1:5000; Abcam; ab98968), and anti- \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\upbeta }$$\end{document} β -actin antibody (1:10,000; Abcam; ab6276) were used.

Techniques: Western Blot, Control

Journal: The Journal of Neuroscience

Article Title: NMDA Receptors Are Upregulated and Trafficked to the Plasma Membrane after Sigma-1 Receptor Activation in the Rat Hippocampus

doi: 10.1523/JNEUROSCI.0458-14.2014

Figure Lengend Snippet: Change in protein expression of NMDARs, AMPARs, and PSD-95 after SKF or PTZ administration

Article Snippet: The following antibodies and their dilutions were used in this study: mouse monoclonal anti-GluN1 (1:10,000; Synaptic Systems); mouse monoclonal anti-GluN2A and mouse monoclonal anti-GluN2B (both 1:750; LifeSpan Biosciences); rabbit monoclonal anti-GluA1 clone C3T (1:2500; Millipore); rabbit polyclonal anti-GluA2/3/4 (1:3500; Cell Signaling Technology); mouse monoclonal anti-PSD-95 (1:10,000; Affinity BioReagents); and goat polyclonal anti-σ-1R (1:250; Santa Cruz Biotechnology).

Techniques: Expressing

Journal: The Journal of Neuroscience

Article Title: NMDA Receptors Are Upregulated and Trafficked to the Plasma Membrane after Sigma-1 Receptor Activation in the Rat Hippocampus

doi: 10.1523/JNEUROSCI.0458-14.2014

Figure Lengend Snippet: Injection of σ-1R agonists leads to an increase in GluN2 subunits and PSD-95 expression. A significant increase in protein expression of GluN2A, GluN2B, and PSD-95 was observed 90 min after intraperitoneal injection of vehicle (Veh) or SKF (A, B). Similar results were obtained 90 min after injection of PRE (C) or PTZ (D). There was no change in the expression level of AMPARs after SKF (E, F), PRE (G), or PTZ (H) injection. Bar graphs are mean ± SEM of at least five animals. *p < 0.05.

Article Snippet: The following antibodies and their dilutions were used in this study: mouse monoclonal anti-GluN1 (1:10,000; Synaptic Systems); mouse monoclonal anti-GluN2A and mouse monoclonal anti-GluN2B (both 1:750; LifeSpan Biosciences); rabbit monoclonal anti-GluA1 clone C3T (1:2500; Millipore); rabbit polyclonal anti-GluA2/3/4 (1:3500; Cell Signaling Technology); mouse monoclonal anti-PSD-95 (1:10,000; Affinity BioReagents); and goat polyclonal anti-σ-1R (1:250; Santa Cruz Biotechnology).

Techniques: Injection, Expressing

Journal: The Journal of Neuroscience

Article Title: NMDA Receptors Are Upregulated and Trafficked to the Plasma Membrane after Sigma-1 Receptor Activation in the Rat Hippocampus

doi: 10.1523/JNEUROSCI.0458-14.2014

Figure Lengend Snippet: The increase in NMDAR subunit and PSD-95 expression is also observed after σ-1R activation in hippocampal slices. A, WBs showing the protein level of GluN1, GluN2A, GluN2B, and PSD-95 after a 90 min bath application of 1 or 5 μm SKF. B, A significant increase in GluN1, GluN2A, and GluN2B expression (relative to control; Ctrl) was observed in the presence of 1 μm SKF. A significant increase in PSD-95 expression was observed after application of 5 μm SKF. C, D, The increase in NMDAR and PSD-95 protein levels was abolished after pretreatment with 50 μm BD1063. Bar graphs are mean ± SEM of slices from at least five animals per treatment.

Article Snippet: The following antibodies and their dilutions were used in this study: mouse monoclonal anti-GluN1 (1:10,000; Synaptic Systems); mouse monoclonal anti-GluN2A and mouse monoclonal anti-GluN2B (both 1:750; LifeSpan Biosciences); rabbit monoclonal anti-GluA1 clone C3T (1:2500; Millipore); rabbit polyclonal anti-GluA2/3/4 (1:3500; Cell Signaling Technology); mouse monoclonal anti-PSD-95 (1:10,000; Affinity BioReagents); and goat polyclonal anti-σ-1R (1:250; Santa Cruz Biotechnology).

Techniques: Expressing, Activation Assay

Journal: The Journal of Neuroscience

Article Title: NMDA Receptors Are Upregulated and Trafficked to the Plasma Membrane after Sigma-1 Receptor Activation in the Rat Hippocampus

doi: 10.1523/JNEUROSCI.0458-14.2014

Figure Lengend Snippet: Both acute and chronic administration of σ-1R antagonists block the increase in GluN2 subunit and PSD-95 observed after SKF, PTZ, or PRE administration

Article Snippet: The following antibodies and their dilutions were used in this study: mouse monoclonal anti-GluN1 (1:10,000; Synaptic Systems); mouse monoclonal anti-GluN2A and mouse monoclonal anti-GluN2B (both 1:750; LifeSpan Biosciences); rabbit monoclonal anti-GluA1 clone C3T (1:2500; Millipore); rabbit polyclonal anti-GluA2/3/4 (1:3500; Cell Signaling Technology); mouse monoclonal anti-PSD-95 (1:10,000; Affinity BioReagents); and goat polyclonal anti-σ-1R (1:250; Santa Cruz Biotechnology).

Techniques: Blocking Assay

Journal: The Journal of Neuroscience

Article Title: NMDA Receptors Are Upregulated and Trafficked to the Plasma Membrane after Sigma-1 Receptor Activation in the Rat Hippocampus

doi: 10.1523/JNEUROSCI.0458-14.2014

Figure Lengend Snippet: Chronic and acute administration of BD1047 abolishes the σ-1R-mediated increase in NMDAR subunit and PSD-95 expression in vivo. A, B, There was no change in the expression level of GluN2A, GluN2B, or PSD-95 after a 2 d chronic administration or a single intraperitoneal injection (acute administration) of vehicle (Veh) or the σ-1R antagonist BD1047 (BD). C, D, Chronic administration of BD1047 blocked the increase in GluN2 subunit and PSD-95 levels observed 90 min after an intraperitoneal injection of SKF or PTZ. E, F, Acute administration of BD1047 also abolished the increase in GluN2 subunits and PSD-95 expression after administration of SKF or PRE. Bar graphs are mean ± SEM of at least five animals.

Article Snippet: The following antibodies and their dilutions were used in this study: mouse monoclonal anti-GluN1 (1:10,000; Synaptic Systems); mouse monoclonal anti-GluN2A and mouse monoclonal anti-GluN2B (both 1:750; LifeSpan Biosciences); rabbit monoclonal anti-GluA1 clone C3T (1:2500; Millipore); rabbit polyclonal anti-GluA2/3/4 (1:3500; Cell Signaling Technology); mouse monoclonal anti-PSD-95 (1:10,000; Affinity BioReagents); and goat polyclonal anti-σ-1R (1:250; Santa Cruz Biotechnology).

Techniques: Expressing, In Vivo, Injection

Journal: The Journal of Neuroscience

Article Title: NMDA Receptors Are Upregulated and Trafficked to the Plasma Membrane after Sigma-1 Receptor Activation in the Rat Hippocampus

doi: 10.1523/JNEUROSCI.0458-14.2014

Figure Lengend Snippet: The increase in GluN2 subunits and PSD-95 expression after σ-1R activation is protein synthesis dependent. A, Representative WBs showing that the expression levels of GluN2A, GluN2B, and PSD-95 were unaffected by intraperitoneal injection of vehicle (Veh) or anisomycin (Aniso). However, there was a decrease in the expression level of c-Fos (B), demonstrating that anisomycin blocked protein synthesis at this time point. Interestingly, anisomycin treatment before SKF (C, D) or PTZ (E, F) administration prevented any change in expression levels of GluN2 subunits and PSD-95. Bar graphs are mean ± SEM of at least five animals. *p < 0.05.

Article Snippet: The following antibodies and their dilutions were used in this study: mouse monoclonal anti-GluN1 (1:10,000; Synaptic Systems); mouse monoclonal anti-GluN2A and mouse monoclonal anti-GluN2B (both 1:750; LifeSpan Biosciences); rabbit monoclonal anti-GluA1 clone C3T (1:2500; Millipore); rabbit polyclonal anti-GluA2/3/4 (1:3500; Cell Signaling Technology); mouse monoclonal anti-PSD-95 (1:10,000; Affinity BioReagents); and goat polyclonal anti-σ-1R (1:250; Santa Cruz Biotechnology).

Techniques: Expressing, Activation Assay, Injection

Journal: The Journal of Neuroscience

Article Title: NMDA Receptors Are Upregulated and Trafficked to the Plasma Membrane after Sigma-1 Receptor Activation in the Rat Hippocampus

doi: 10.1523/JNEUROSCI.0458-14.2014

Figure Lengend Snippet: An increase in interaction between σ-1Rs and NMDARs was observed after SKF injection. A, Representative blots showing the specificity of the σ-1R antibody. Single σ-1Rs band detected in rat brain homogenate was abolished in the presence of blocking peptide (A, left). Similarly, the single σ-1Rs band in wild-type mouse hippocampus was not detected in σ-1R−/− mice (A, right). B, Control Co-IP experiment demonstrating that σ-1Rs were immunoprecipitated and detected with the σ-1R antibody. C, Representative WBs from a Co-IP experiment investigating the interaction between σ-1Rs and GluN2 subunits, demonstrating an increased interaction after SKF administration compared with vehicle (Veh). D, Pooled data showing a significant increase in interaction between σ-1Rs and GluN2 subunits after SKF injection. E, Representative WBs from an additional experiment investigating the interaction between PSD-95 and GluN2 subunits after SKF administration. F, The binding of PSD-95 with GluN2A was unaffected by SKF; interestingly, a significant decrease in the interaction between PSD-95 and GluN2B was observed. Bar graphs are mean ± SEM of at least five animals. *p < 0.05.

Article Snippet: The following antibodies and their dilutions were used in this study: mouse monoclonal anti-GluN1 (1:10,000; Synaptic Systems); mouse monoclonal anti-GluN2A and mouse monoclonal anti-GluN2B (both 1:750; LifeSpan Biosciences); rabbit monoclonal anti-GluA1 clone C3T (1:2500; Millipore); rabbit polyclonal anti-GluA2/3/4 (1:3500; Cell Signaling Technology); mouse monoclonal anti-PSD-95 (1:10,000; Affinity BioReagents); and goat polyclonal anti-σ-1R (1:250; Santa Cruz Biotechnology).

Techniques: Injection, Blocking Assay, Co-Immunoprecipitation Assay, Immunoprecipitation, Binding Assay

Journal: The Journal of Neuroscience

Article Title: NMDA Receptors Are Upregulated and Trafficked to the Plasma Membrane after Sigma-1 Receptor Activation in the Rat Hippocampus

doi: 10.1523/JNEUROSCI.0458-14.2014

Figure Lengend Snippet: A redistribution of GluN2 subunits and PSD-95 was observed after administration of SKF. A, Schematic diagram showing the three fractions isolated from LP1 by discontinuous sucrose gradient. B, Representative WBs showing protein expression in the different fractions after injection of vehicle (Veh) or SKF with β-tubulin as a loading control. A significant increase in GluN2A (C) and GluN2B (D) expression was observed in the VF and a significant increase in PSD-95 (E) in both VF and SMF after SKF administration. There is a decrease in expression in the PF for all three proteins investigated (C–E). Bar graphs are mean ± SEM of at least four animals. *p < 0.05.

Article Snippet: The following antibodies and their dilutions were used in this study: mouse monoclonal anti-GluN1 (1:10,000; Synaptic Systems); mouse monoclonal anti-GluN2A and mouse monoclonal anti-GluN2B (both 1:750; LifeSpan Biosciences); rabbit monoclonal anti-GluA1 clone C3T (1:2500; Millipore); rabbit polyclonal anti-GluA2/3/4 (1:3500; Cell Signaling Technology); mouse monoclonal anti-PSD-95 (1:10,000; Affinity BioReagents); and goat polyclonal anti-σ-1R (1:250; Santa Cruz Biotechnology).

Techniques: Isolation, Expressing, Injection