Journal: bioRxiv

Article Title: Synaptic accumulation of GluN2B-containing NMDA receptors mediates the effects of BDNF-TrkB signalling on synaptic plasticity and in epileptogenesis

doi: 10.1101/2024.10.21.618702

Figure Lengend Snippet: ( A ) Representative images of hippocampal synaptoneurosomes (prepared from adult Sprague-Dawley rats 6-8 weeks old) incubated with BDNF (50 ng/mL). Synaptoneurosomes were immunoassayed for GluN2B using an antibody against an extracellular epitope in the GluN2B N-terminus and immunoassayed for vGlut1, PSD-95. Merge scale bar, 10 μm. Insert scale bar, 0.5 µm. Images illustrated in ( A ) were analyzed for the GluN2B integrated density ( B ) and GluN2B mean gray value ( C ). Data are the means ± SEM of 779 - 840 synaptoneurosomes per condition, in at least three independent experiments performed in different preparations. ****p < 0.0001, by Kruskal-Wallis’s test and Dunn’s multiple comparisons test.

Article Snippet: To label surface GluN2B-containing NMDAR, live neurons (low-density hippocampal cultures) were incubated for 10 min at room temperature (RT) with an antibody against an extracellular epitope of the GluN2B N-terminus (1:100; AGC-003, Alomone Labs) diluted in a saline buffer (145 mM NaCl, 5 mM glucose, 10 mM HEPES, 5 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2, [pH 7.3]) (Sham) solution, as previously described ( ).

Techniques: Incubation

Journal: bioRxiv

Article Title: Synaptic accumulation of GluN2B-containing NMDA receptors mediates the effects of BDNF-TrkB signalling on synaptic plasticity and in epileptogenesis

doi: 10.1101/2024.10.21.618702

Figure Lengend Snippet: ( A ) Representative images of hippocampal neurons (DIV 14 - 15) that were stimulated with BDNF (50 ng/ml for 10 or 30 min), as indicated. Neurons were then live-immunoassayed for GluN2B using an antibody against an extracellular epitope in the GluN2B N-terminus, fixed, and then further immunoassayed for PSD-95, vGlut1, and MAP2. Scale bar, 5 μm. Images illustrated in ( A) , were analyzed for the total number ( B ), intensity ( C ), and area ( D ) of surface GluN2B puncta per µm. Synaptic (PSD-95- and vGlut1-colocalized) surface GluN2B number ( E ), area ( F ), and intensity ( G ) of puncta per density of excitatory synapses (number of puncta PSD-95–vGlut1 colocalized per µm), were also analyzed. Data are normalized to the means of the control and are the means ± SEM of 43-45 cells per condition, from at least three independent experiments performed in different preparations. *p < 0.05, **p < 0.01 by one-way analysis of variance (ANOVA) followed by Bonferroni post-test.

Article Snippet: To label surface GluN2B-containing NMDAR, live neurons (low-density hippocampal cultures) were incubated for 10 min at room temperature (RT) with an antibody against an extracellular epitope of the GluN2B N-terminus (1:100; AGC-003, Alomone Labs) diluted in a saline buffer (145 mM NaCl, 5 mM glucose, 10 mM HEPES, 5 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2, [pH 7.3]) (Sham) solution, as previously described ( ).

Techniques: Control

Journal: bioRxiv

Article Title: Synaptic accumulation of GluN2B-containing NMDA receptors mediates the effects of BDNF-TrkB signalling on synaptic plasticity and in epileptogenesis

doi: 10.1101/2024.10.21.618702

Figure Lengend Snippet: ( A ) Representative images of hippocampal neurons (DIV 14 - 15) pre-incubated with GÖ 6983 (100 nM) or vehicle (DMSO; 1:1000 dilution for 40 min) and then either maintained under the same conditions or stimulated with BDNF (50 ng/ml for 30 min), as indicated. Neurons were live-immunoassayed for GluN2B using an antibody against an extracellular epitope in the GluN2B N-terminus, fixed, and then further immunoassayed for PSD-95, vGlut1, and MAP2. Scale bar, 5 μm. Images illustrated in ( A ) were analyzed for the total number ( B ), area ( C ), and intensity ( D ) of surface GluN2B puncta per µm. Synaptic (PSD-95- and vGlut1-colocalized) surface GluN2B number ( E ), area ( F ), and intensity ( G ) of puncta per µm of excitatory synapses (number of puncta PSD-95–vGlut1 colocalized per µm), were also analyzed. Data are normalized to the mean of the DMSO control and are the means ± SEM of 28 - 30 cells per condition, in at least three independent experiments performed in different preparations. ***p < 0.001, ****p < 0.0001 by one-way analysis of variance (ANOVA) followed by Bonferroni post-test.

Article Snippet: To label surface GluN2B-containing NMDAR, live neurons (low-density hippocampal cultures) were incubated for 10 min at room temperature (RT) with an antibody against an extracellular epitope of the GluN2B N-terminus (1:100; AGC-003, Alomone Labs) diluted in a saline buffer (145 mM NaCl, 5 mM glucose, 10 mM HEPES, 5 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2, [pH 7.3]) (Sham) solution, as previously described ( ).

Techniques: Incubation, Control

Journal: bioRxiv

Article Title: Synaptic accumulation of GluN2B-containing NMDA receptors mediates the effects of BDNF-TrkB signalling on synaptic plasticity and in epileptogenesis

doi: 10.1101/2024.10.21.618702

Figure Lengend Snippet: ( A ) Experimental design for the lithium-pilocarpine model of Status Epilepticus. ( B ) Representative images of hippocampal synaptoneurosomes (prepared from adult Sprague-Dawley rats 6-8 weeks old, treated with saline, saline and ANA-12, Pilocarpine or Pilocarpine and ANA-12). Synaptoneurosomes were live-immunoassayed for GluN2B using an antibody against an extracellular epitope in the GluN2B N-terminus and immunoassayed for vGlut1 and PSD-95. Merge scale bar, 10 μm. Insert scale bar, 0.5 µm. Images illustrated in ( B ) were analyzed for the GluN2B integrated density ( C ) and GluN2B mean gray value ( D ). Data are the means ± SEM of 779 - 840 synaptoneurosomes per condition, from at least four animals for each experimental condition. ****p < 0.0001, **p<0.01 as determined by Kruskal Wallis’s test and Dunn’s multiple comparisons test. Representative western blot and analysis ( E-G ). Proteins were extracted from synaptoneurosomes prepared from the same animals used in the immunocytochemistry experiments. For the immunoblot, antibodies against pTrkB, total TrKB and β-Tubulin were used. In this analysis, pTrkB levels were normalized to total TrkB levels and total TrkB levels were normalized to β-tubulin. Data are means ± SEM of at least four animals for each experimental condition. *p < 0.05, by one-way analysis of variance (ANOVA) followed by Dunnett’s post-test.

Article Snippet: To label surface GluN2B-containing NMDAR, live neurons (low-density hippocampal cultures) were incubated for 10 min at room temperature (RT) with an antibody against an extracellular epitope of the GluN2B N-terminus (1:100; AGC-003, Alomone Labs) diluted in a saline buffer (145 mM NaCl, 5 mM glucose, 10 mM HEPES, 5 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2, [pH 7.3]) (Sham) solution, as previously described ( ).

Techniques: Saline, Western Blot, Immunocytochemistry

Journal: Health

Article Title: Long-Term Impact of Maternal Protein Malnutrition on Learning and Memory Abilities and DNA Methylating Profiles of the Nervous System in Offspring Rats

doi: 10.4236/health.2014.615239

Figure Lengend Snippet: Figure 6. Protein expressions in 8-week-old offspring rats’ brain tissue. (a)-(e) show Western blotting and immunohisto- chemical results for NMDAR1, NMDAR2B, DR1, DR2 and GR; (f) The reference factor was the data gathered on the five types of target gene mRNAs of the control group. demethylation within mature neurons and suppresses synaptic function [23]. Thus, several changes in methyla- tion in the hippocampus of 8-week-old offspring rats was found, and had an effect on the methylation status of the Grin2b promoter; it also produced a decrease in the expression of NMDAR2B in offspring, which played an important role in the LTP effect. The LTP effect is the basis of learning and memory in the hippocampus, the key structure in controlling spatial orientation [24] [25], which is susceptible to damage under acute and chronic stress [26]. The embryonic and fetal period is the most vulnerable stage during pregnancy. It was speculated that thein utero effect on the hippocampus might be the early stage of cognitive and learning dysfunction of offspring, as previous studies have shown that this anatomical abnormality may lead to deficits in hippocampal-related behaviors [27]-[29]. The LTP effect involved histone modification and DNA methylation [30]-[33]. Additional- ly, the key receptors, NMDA receptors, are involved in a wide range of learning and memory activities, synaptic plasticity, neural development, ischemic brain injury, neurodegeneration, epilepsy, cancer and many other im- portant physiological and pathological processes [34]-[37]. Also, there was an adverse impact on the learning pattern in both 8-week-old and 1-year-old offspring, ac- companied by a lower birth weight associated with catch-up growth, increased serum glucose level, reduced se- rum corticosterone and ACTH levels of offspring, and morphological changes in cerebral histiocytes, including the fetal hippocampus, similar to the findings of human epidemiology studies [38]-[40].

Article Snippet: Dropped antibody NMDAR1, NMDAR2B, DR1 (Wuhan Boster Company BA0612, BA0614, BA08031:100), DR2 (Wuhan Boster Company BA0804 1:20), GR (Wuhan Boster Company BA0895 1:40) were diluted by Triton-x-100 TBS (PH = 7) and applied onto the sections, with TBS as a negative control.

Techniques: Western Blot, Control, Methylation, Produced, Expressing, Modification, DNA Methylation Assay

Journal: iScience

Article Title: Distinct effects of AMPAR subunit depletion on spatial memory

doi: 10.1016/j.isci.2023.108116

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-NMDAR2B antibody (anti-GluN2B) , Fisher Scientific , Cat# AB1557.

Techniques: Recombinant, Microscopy, Protease Inhibitor, Western Blot, Bicinchoninic Acid Protein Assay, Mutagenesis, Imaging, Software

Journal: Scientific Reports

Article Title: New Alzheimer’s disease model mouse specialized for analyzing the function and toxicity of intraneuronal Amyloid β oligomers

doi: 10.1038/s41598-019-53415-8

Figure Lengend Snippet: Immunoblot analysis of expression ratios of the pre and postsynaptic proteins in Aβ-GFP Tg and non-Tg mice synapse. ( A,B ) Synaptosome fractions from hippocampus (20 μg/lane) were immunoblotted against presynaptic markers ( A ; synaptophysin, VAMP-2, syntaxin-1, and Munc13–1) and postsynaptic markers ( B ; PSD-95, GluN1, GluN2A, GluN2B, GluA2, and neuroligin). We prepared 4 sets of Aβ-GFP Tg/non-Tg samples. Band intensities of Aβ-GFP Tg mice were normalized by those of actin and the expression were calculated ratios with the values of non-Tg mice as 1. There was no difference in expression ratios of the presynaptic proteins we examined. However, expression of the postsynaptic proteins GluN2B, Glu2A, and neuroligin were significantly decreased in Aβ-GFP Tg mice at 3-month-old (*p < 0.05 by Mann-Whitney’s U test, n = 4 that included hippocampi from 3 animals each, data are presented as means ± SEM). ( C ) The expression ratios of GluN2B in homogenate. There was no significant difference in the expression ratios of GluN2B in homogenates of Aβ-GFP Tg and non-Tg mice (Mann-Whitney’s U test, n = 5 animals each, data are presented as means ± SEM).

Article Snippet: VAMP-2 (1:5000, Abcam, Cambridge, UK), Munc13-1(1:1000, Synaptic Systems, Gottingen, Germany), syntaxin-1 (1:1000, Merck), synaptophysin (1:2000, Sigma-Aldrich), PSD-95 (1:200, Merck), GluN1 (1:200, Merck), GluN2A (1:500, Merck), GluN2B (1:200, Merck), GluA2 (1:200, Merck), neuroligin 1(1:500, Synaptic Systems), and actin (1:2000, Merck) antibodies were used to probe the corresponding proteins and ECL was used for detection (Immunostar Zeta and Immunostar LD, FUJIFILM Wako Pure Chemical Corporation).

Techniques: Western Blot, Expressing