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: Cognitive Neurodynamics

Article Title: U1 snRNA over-expression affects neural oscillations and short-term memory deficits in mice

doi: 10.1007/s11571-019-09528-x

Figure Lengend Snippet: a The representative immune reactive bands of NR2A, NR2B, GABAARα1, GAD67 and β-actin of the CON and U1OE mice. b A comparison of the optical density ratio of NR2A/β-actin between CON group and U1OE group. c A comparison of the optical density ratio of NR2B/β-actin between the U1OE group and the CON group. d A comparison of the optical density ratio of GABAARα1/β-actin between the U1OE group and the CON group. e A comparison of the optical density ratio of GAD67/β-actin between the U1OE group and the CON group. f Correlations between the level of neurons excitatory proteins (NR2B) and recognition index of NORT. g Correlations between neuron inhibitory proteins (GABBARα1) and recognition index of NORT. Data are expressed as mean ± SEM. ***p < 0.001 comparison between the CON group and the U1OE group

Article Snippet: Rabbit polyclonal antibodies to NR2A (Abcam, 1:2000), NR2B (Abcam, 1:2000) and GAD67 (Abcam, 1:2000), mouse polyclonal antibody to GABAAR α1 (Abcam, 1:2000).

Techniques:

Journal: Hippocampus

Article Title: Preferential frequency-dependent induction of synaptic depression by the lateral perforant path and of synaptic potentiation by the medial perforant path inputs to the dentate gyrus.

doi: 10.1002/hipo.23338

Figure Lengend Snippet: FIGURE 11 NMDA receptor expression does not differ in the middle and outer thirds of the molecular layer of the dentate gyrus. (a–c) Immunohistochemically stained sections are shown that were used to assess the expression of GluN1, GluN2A, and GluN2B subunits of the NMDA receptor in the dorsal hippocampus. The laminar structure of the hippocampal DG, as well as pale, receptor-devoid regions of the brain sections are visible. The inner (suprapyramidal) blade of the DG was scrutinized, as this was the region from which electrophysiological recordings were made. Of this, the outer and middle thirds of the molecular layer were assessed, as these are the components of the molecular layer where the lateral perforant path (LPP) and medial perforant path (MPP) form synapses, respectively. (o. ml, outer molecular layer; m. ml, middle molecular layer). The bar charts describe the (averaged) expression of GluN1, GluN2A, and GluN2B subunits of the NMDA receptor in the outer and middle molecular layers of the inner/suprapyramidal blade of the dentate gyrus. Subunit expression is not statistically different in the outer compared to the middle molecular layers. Open circles in the bar charts represent the expression levels of each individual animal, the averaged data from which is represented by the bar

Article Snippet: Here, the sections were incubated for 5 days at 4 C with primary antibody solutions: mouse monoclonal anti-NMDAR1 (GluN1) (556308, PharMingen, Becton, Dickinson and Company, Franklin Lakes, NJ) at 1:200 dilution, or rabbit polyclonal anti-NMDAε1 (GluN2A) (sc-9056, Santa Cruz Biotechnology) 1:750 in a dilution medium containing 1% BSA in 0.2% tris-buffered saline (TBS)-Tx.

Techniques: Expressing, Staining

Journal: Frontiers in Endocrinology

Article Title: Dorzagliatin shows potential in preventing cognitive impairment in diabetes: evidence from Mendelian randomization analysis and animal study

doi: 10.3389/fendo.2025.1755359

Figure Lengend Snippet: Dorzagliatin prevented diabetes-induced downregulation of synaptic proteins in Goto Kakizaki rats. (A) Western blot analysis of selected glutamate receptors and postsynaptic density protein 95 (PSD-95) in hippocampus of each group. GAPDH was used as an internal control. (B–D) Statistics of GluN1 (B) , GluN2A (C) and PSD-95 (D) protein levels in the hippocampus of T2D (Goto Kakizaki-vehicle) and control (Wistar-vehicle). (E) Western blot analysis of selected glutamate receptors and postsynaptic density protein 95 (PSD-95) in the hippocampus of Goto Kakizaki-vehicle and Goto Kakizaki-dorzagliatin rats. (F–H) Statistics of GluN1 (F) , GluN2A (G) and PSD-95 (H) protein levels in the hippocampus of Goto Kakizaki-vehicle group and Goto Kakizaki-dorzagliatin group. Data are expressed as mean ± SEM. Student’s t test, two tailed. *P < 0.05, **P < 0.01. (n=4 per group). hippo, hippocampus; dorza, dorzagliatin.

Article Snippet: The membranes were blocked with 1×TBST and 5% BSA (tank blotting) or 5% skim milk (Semi-dry blotting) for 1 hour at room temperature and then incubated overnight at 4°C with following primary antibodies, respectively: mouse monoclonal anti-GluN1 antibody (Millipore, Cat. No: 05-432); rabbit polyclonal anti-GluN2A antibody (NOVUS, Cat. No: NB300-105); rabbit monoclonal anti-GLUT1 antibody (Abcam, Cat. No: ab115730); rabbit polyclonal anti-GLUT3 antibody (Bioss, Cat. No: bs-1207R); rabbit polyclonal anti-IR antibody (Abcam, Cat. No: ab137747); rabbit monoclonal anti-PSD95 antibody (Abcam, Cat. No: ab238135).

Techniques: Western Blot, Control, Two Tailed Test