Journal: Annals of neurology

Article Title: Absence of Neuronal Autoantibodies in Neuropsychiatric Systemic Lupus Erythematosus.

doi: 10.1002/ana.25908

Figure Lengend Snippet: Figure 1. Clinical features and immunoglobulin G (IgG) binding to conformational neuronal surface epitopes from plasma and cerebrospinal fluid (CSF) of patients with neuropsychiatric lupus (NPSLE). A. Clinical features of 35 patients with SLE, including 15 with active NPSLE at the time of sampling. 32/34 (94%) were on immunotherapies, including prednisolone (71%), hydroxychloroquine (41%), belimumab (6%) and rituximab (3%). ANA = anti-nuclear antibody. B. Live cell-based assays employing HEK293T cells with surface expressed N-methyl D-aspartate receptors (NMDARs) as NR1-NR2A or NR1-NR2B heteromers (nuclei highlighted with DAPI, 4′,6-diamidino-2-phenylindole). First panel shows that a commercial antibody directed against the extracellular domain of the NR2A-subunit (red) binds to HEK293T cells which express NR1-NR2A heteromers. NMDAR-antibody encephalitis (NMDAR-Ab-E) patient serum IgGs (n=11) bind to the NR1-NR2A heteromers (second panel), and to NR1-NR2B heteromers / NR1 homomers (data not shown). NMDAR-antibody patient CSFs show similar binding (as Irani et al, 2010).10 Serum from healthy controls (n=36), plasma and CSFs from SLE patients (n=35 of each) show no binding. No binding was observed with the G11 antibody (data not shown, lower panel representative). Throughout, IgG binding was visualized with an anti-human 568-Alexaflour antibody (1:750 dilution, A-21090) C. Commercial antibodies against the extracellular domains of NR1 (Alomone, AGC-001), NR2A (Alomone,

Article Snippet: Throughout, IgG binding was visualized with an anti-human 568-Alexaflour antibody (1:750 dilution, A-21090) C. Commercial antibodies against the extracellular domains of NR1 (Alomone, AGC-001), NR2A (Alomone, AGC-002) and NR2B (kind gift from Prof Stephenson, London) subunits bound to the surface of live HEK293T cells co-transfected with EGFP plus NR1 and NR2B subunits.

Techniques: Binding Assay, Clinical Proteomics, Sampling

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