Journal: Journal of Neurochemistry

Article Title: Pharmacological target sites for restoration of age‐associated deficits in NMDA receptor‐mediated norepinephrine release in brain

doi: 10.1111/jnc.16280

Figure Lengend Snippet: Characterizing the cellular localization of the NMDA receptors regulating [ 3 H] ‐NE release in young rat cortical brain slices. 1 mM Glu‐stimulated‐[ 3 H]‐NE releases in the cerebral cortex tissue slices from young rats ( n = 3–7) in the presence 1 & 3 μM of the TTX, voltage‐gated Na channel blocker, 10 μM MK‐801, and a combination of MK‐801 and TTX. Data are expressed as mean (±SEM) of net fractional release (stimulated—basal), with each data point representing a duplicate from one animal. Data were analyzed using a mixed‐effect analysis followed by Dunnett's multiple comparison test **** p < 0.0001. NE, norepinephrine; Glu, glutamate; TTX, tetrodotoxin.

Article Snippet: The NMDA receptor NR2A and NR2B subunit polyclonal antibodies were used alongside the NR1 monoclonal antibody (PhosphoSolutions, CO, USA; Cat # 1805‐NR1).

Techniques: Comparison

Journal: Journal of Neurochemistry

Article Title: Pharmacological target sites for restoration of age‐associated deficits in NMDA receptor‐mediated norepinephrine release in brain

doi: 10.1111/jnc.16280

Figure Lengend Snippet: The stimulatory effect of Glutamate Vs. NMDA on [ 3 H] ‐NE release in young rat cortical brain slices. In (a), the concentrations‐response curves of glutamate and NMDA‐stimulated [ 3 H] ‐NE releases in the cerebral cortex tissue slices from young rats ( n = 4). In (b), the 1 mM glutamate and NMDA stimulated NE release in the presence and absence of 1.2 mM magnesium in the cerebral cortex tissue slices from young rats ( n = 3). Data were analyzed using an unpaired t ‐test. * p ≤ 0.05, ** p ≤ 0.01 NE, norepinephrine; Glu, glutamate; NMDA, N‐methyl‐ d ‐aspartate; Mg 2+ , magnesium.

Article Snippet: The NMDA receptor NR2A and NR2B subunit polyclonal antibodies were used alongside the NR1 monoclonal antibody (PhosphoSolutions, CO, USA; Cat # 1805‐NR1).

Techniques:

Journal: Journal of Neurochemistry

Article Title: Pharmacological target sites for restoration of age‐associated deficits in NMDA receptor‐mediated norepinephrine release in brain

doi: 10.1111/jnc.16280

Figure Lengend Snippet: Age‐associated changes in the expression of NMDA receptors freely solubilize subunits in the cortical rat tissue homogenate. A representative Western blot for NMDA receptors subunits in the cerebral cortex in (a); and in (b) quantified results for GluN1, GluN2A, and GluN2B expressions in young and aged rats ( n = 5). Data are expressed as mean (±SEM) normalized to young rats, with each data point representing a duplicate from one animal. Data were analyzed using an unpaired t ‐test. * p ≤ 0.05. NMDA, N‐methyl‐ d ‐aspartate).

Article Snippet: The NMDA receptor NR2A and NR2B subunit polyclonal antibodies were used alongside the NR1 monoclonal antibody (PhosphoSolutions, CO, USA; Cat # 1805‐NR1).

Techniques: Expressing, Western Blot

Journal: Journal of Neurochemistry

Article Title: Pharmacological target sites for restoration of age‐associated deficits in NMDA receptor‐mediated norepinephrine release in brain

doi: 10.1111/jnc.16280

Figure Lengend Snippet: Effect of aging on [ 3 H]‐MK‐801 binding to NMDA receptors in the young (2–3 months old) and aged (18–24 months old) rat cortical tissue membrane. In (a) saturation curve of [ 3 H]‐MK‐801 binding to NMDA receptors in young rats in the presence of 10 μM Glu and Gly ( n = 4). The non‐linear least‐squares fitting of the saturation isotherm yielded K d and B max values of 1.8 nM and 970 fmol/mg of protein, respectively. Both total and non‐specific binding of [ 3 H]‐MK‐801 is shown in the curve, and the specific [ 3 H]‐MK‐801 binding is presented with 95% CI in dotted lines. Inset: Saturation data graphed as Scatchard plots. Whereas in (b), the binding of 10 nM [ 3 H]‐MK‐801 in +/− 10 μM Glu and Gly in young and aged rats ( n = 7), each performed in triplicate and repeated twice. In (c), the % increases after subtracting baseline binding from the binding in the presence of 10 μM Glu and Gly. Baseline Binding values represent [ 3 H]‐MK‐801 binding without exogenous addition of Glu and Gly. Data were analyzed using a Mixed‐effect analysis followed by Tukey's multiple comparison tests. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001; whereas the % of increase over the baseline was analyzed using an unpaired t ‐test: ** p ≤ 0.01. NMDA, N‐methyl‐d‐aspartate; Glu, Glutamate; Gly, Glycine; K d , dissociation constant; B max , Maximum Binding; n H , Hill Coefficient).

Article Snippet: The NMDA receptor NR2A and NR2B subunit polyclonal antibodies were used alongside the NR1 monoclonal antibody (PhosphoSolutions, CO, USA; Cat # 1805‐NR1).

Techniques: Binding Assay, Membrane, Comparison

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: Molecular Neurodegeneration

Article Title: Genetic ablation of dynactin p150 Glued in postnatal neurons causes preferential degeneration of spinal motor neurons in aged mice

doi: 10.1186/s13024-018-0242-z

Figure Lengend Snippet: P150 Glued deficiency increases protein expression of glutamate receptors and vulnerability to glutamate-mediated excitotoxicity in spinal neurons. a Western blot analysis shows the expression of AMPAR subunits GLUR1 and GLUR2, NMDAR subunits NR2A and NR2B, postsynaptic density protein 95 (PSD95) and synaptophysin (SYP) in the spinal cord homogenate of 9-month-old Dctn1 LoxP/LoxP and Dctn1 LoxP/LoxP ; Thy1-Cre mice. Actin was used as loading control. b Bar graphs show the quantification of GLUR1, GLUR2, NR2A, NR2B, PSD95 and SYP level normalized with actin (n = 10 per genotype). Data were presented as mean ± SEM; ** p < 0.01, *** p < 0.001. Unpaired t-test was used for statistical analysis. c Western blot analysis shows the levels of biotinylated cell surface and total AMPAR subunits GLUR1 and GLUR2, NMDAR subunits NR2A and NR2B in cultured Dctn1 LoxP/LoxP and Dctn1 LoxP/LoxP ; Cre/Esr1 spinal neurons at 21 DIV. Actin was used as loading control. d Bar graphs show the quantification of total and surface GLUR1, GLUR2, NR2A and NR2B level normalized with actin, and the ratio of GLUR1, GLUR2, NR2A and NR2B at cell surface versus total proteins (n = 8 per genotype). Data were presented as mean ± SEM; ** p < 0.01, *** p < 0.001. Unpaired t-test was used for statistical analysis. (E) Primary spinal neurons were treated with glutamate, and the viability of these neurons was measured by the MTT assay. Box & whiskers graphs show survival rate (percentage) of cultured Dctn1 LoxP/LoxP and Dctn1 LoxP/LoxP ; Cre/Esr1 spinal neurons (21 DIV) treated with 0 or 10 μM glutamate for 24 h ( N = 40 per genotype per condition). Data were presented as mean ± SEM. One-way ANOVA plus Tukey’s post hoc test was used for statistical analysis. *** p < 0.001 for difference between Dctn1 LoxP/LoxP neurons treated with 0 and 10 μM glutamate, *** p < 0.001 for difference between Dctn1 LoxP/LoxP ; Cre/Esr1 neurons treated with 0 and 10 μM glutamate, ** p < 0.01 for difference between Dctn1 LoxP/LoxP and Dctn1 LoxP/LoxP ; Cre/Esr1 neurons treated with 10 μM glutamate, while no difference was found between Dctn1 LoxP/LoxP and Dctn1 LoxP/LoxP ; Cre/Esr1 neurons treated with 0 μM glutamate

Article Snippet: The antibodies used for western blot analysis included p150 Glued N-terminus (amino acid 3–202, 1:1000, BD Biosciences), p150 Glued C-terminus (amino acid 1266–1278, 1:1000, Abcam), dynactin subunit p62 (1:1000, Abcam), dynactin subunit p50 (1:5000, BD Biosciences), dynactin subunit Actin Related Protein 1 (ARP1, 1:1000, Sigma-Aldrich), dynein heavy chain (DHC, 1:500, Santa Cruz Biotechnology), dynein intermediate chain (DIC, 1:1000, Sigma-Aldrich), dynein light chain (DLC, 1:500, Santa Cruz Biotechnology), α-tubulin (TUBA, 1:10000, Abcam), acetylated α-tubulin (ac-TUBA, 1:10000, Abcam), tyrosinated α-tubulin (tyro-TUBA, 1:10000, Abcam), detyrosinated α-tubulin (detyro-TUBA, 1:10000, Abcam), Autophagy Related Protein ATG3 (ATG3, 1:1000, Cell Signaling), ATG5 (1:1000, Cell Signaling), ATG7 (1:1000, Cell Signaling), autophagy related protein LC3 (1:1000, Abcam), Lysosomal Associated Membrane Protein 1 (LAMP1, 1:1000, BD Biosciences), LAMP2 (1:1000, Abcam), synaptophysin (SYP, 1:10000, Millipore), Postsynaptic Density Protein 95 (PSD95, 1:1000, Sigma-Aldrich), AMPA receptor subunit GLUR1 (1:1000, Abcam), GLUR2 (1:1000, BD Biosciences), NMDA receptor subunit NR2A (1:250, BD Biosciences), NR2B ((1:500, BD Biosciences), and β-actin (1:5000, Sigma-Aldrich).

Techniques: Expressing, Western Blot, Cell Culture, MTT Assay

Journal: Journal of Neurotrauma

Article Title: Environmental Enrichment Mitigates Deficits after Repetitive Mild Traumatic Brain Injury

doi: 10.1089/neu.2016.4823

Figure Lengend Snippet: Synaptic NR2B expression after TBI. NR2B expression was detected in synaptosome fractions isolated from the cortex and hippocampus 3 days (3d) or 5 weeks (5w) after last injury using western blot. (A) Representative images of NR2B and Na/K-ATPase expression. (B) Densitometric analysis of NR2B expression 3 days after TBI and (C) densitometric analysis of NR2B expression 5 weeks after TBI. NR2B expression was normalized by Na/K-ATPase expression (*p < 0.05, NC-sham vs. NC-TBI; #p < 0.05, NC-TBI vs. EE-TBI). EE, environmental enrichment; NR2B, N-methyl d-aspartate receptor subtype 2B; Na/K-ATPase, sodium potassium ATPase; NC, normal condition; TBI, repetitive mild traumatic brain injury.

Article Snippet: After blocking, proteins were incubated overnight at 4°C with antibodies targeting proteins glutamate receptor 1 (GluR1; 1: 1000; Cell Signaling Technology, Boston, MA), phosphorylated GluR1 (p-GluR1; [s845]; 1:1000; Cell Signaling Technology), p-GluR1 (s831; 1:1000; Abcam, Cambridge, MA), β-actin (1:5000; Abcam), nuclear receptor 1 (NR1; 1:1000; Abcam), N-methyl- d -aspartate (NMDA) receptor subunit 2A (NR2A; 1:1000; Millipore, Billerica, MA), NMDA receptor subunit 2B (NR2B; 1:1000; Abcam), amyloid precursor protein (APP; 1:1000; Millipore), phosphorylated Ca 2+ /calmodulin-dependent protein kinase II (p-CaMKII; 1:2000; Cell signaling Technology), calpain-1 (1:1000; Abcam, Cambridge, UK), postsynaptic density protein 95 (PSD-95; 1:1000; Cell Signaling Technology), and sodium potassium ATPaes (Na/K-ATPase; 1:1000; Millipore).

Techniques: Expressing, Isolation, Western Blot