Journal: bioRxiv

Article Title: GluN2B-mediated regulation of silent synapses for receptor specification and addiction memory

doi: 10.1101/2024.05.19.594887

Figure Lengend Snippet: Homeostatic enrichment of GluN2C-NMDARs in the absence of GluN2B. (A) Example traces of normalized NMDAR-EPSCs (red traces for cKD; black traces for control, left). Averaged NMDAR-EPSCs from D1-MSNs are compared between cocaine-treated mice that received either GluN2B cKD or control virus (n = 33 cells for cKD; n = 25 cells for control groups, right). ( B) I-V curves of normalized NMDAR-EPSCs under 100 µM of Mg 2+ from D1-MSNs in cocaine-treated mice (red, n =18 cells for cKD; black, n = 15 cells for control groups). ( C) averaged traces of NMDAR-EPSCs from cocaine-treated D1-MSNs with GluN2B antagonist (Ifenprodil, magenta) and additional GluN2C antagonist (PPDA, green) at +50 mV holding potential in the presence of PTX and NBQX (left). Sensitivity of NMDAR-EPSCs to Ifenprodil or PPDA is compared between mice that previously received shGluN2B or control virus (n = 10 cells for Ifenprodil/control; n = 9 cells for PPDA/control; n =13 cells for Ifenprodil/cKD; n = 13 cells for PPDA/cKD, right). ( D) Representative confocal images of GluN2C-positive puncta (red) in cocaine-treated D1-MSNs (green, left). Scale bar = 5 μm. Quantification of GluN2C puncta after cocaine administration is compared between mice that received either shGluN2B or control virus (n = 12 cells for cKD; n = 16 cells for control groups, right). ( E) Immuno-electron microscopic images showing subcellular localization of GluN2C (18 nm gold particles, cyan blue arrows) and eYFP for labeling of D1-MSNs (6 nm gold particles, green arrows, left). Scale bar = 100 μm. GluN2C particles present within the synaptic areas are compared between cocaine-treated mice that previously received shGluN2B or control virus (n = 7 cells for both cKD and control groups, right). ( F) Example plots of evoked EPSCs with minimal optical stimulation (failure trials in red dots; successful trials in black dots) without and with PPDA (left). The proportion of silent synapses is compared before and after PPDA administration in cocaine-treated mice that previously received shGluN2B virus (n = 11 cells, right). ( G) An experimental timeline of PPDA or vehicle treatment for behavioral assays. ( H) Quantified CPP scores in the cocaine-paired chamber are compared between PPDA- and vehicle-infused cKD mice (n = 4 mice for PPDA; n = 6 mice for vehicle). ( I) Locomotor activity was monitored upon cocaine infusion on a daily basis between PPDA- and vehicle-treated cKD mice (n = 4 mice for PPDA; n = 5 mice for vehicle groups). Data are represented as mean ± SEM (error bars); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by two-tailed unpaired t test, Mann-whitney, Wilcoxon test, or two-way analysis of variance (ANOVA, with Sidak’s multiple comparisons).

Article Snippet: Sliced sections were blocked with 4 % normal goat serum and 0.45 % Triton X-100 in 0.1 M of phosphate buffer at 4°C for 1 hour and then were incubated with primary antibodies as follows: rabbit anti-GluN2B (AGC-003, Alomone labs., Israel), rabbit anti-GluN2C (AGC-018, Alomone labs.), mouse anti-GluA2 (MAB397, Millipore, MA), rabbit anti-Rac1 (05-389, 1:500, Millipore, MA), or mouse anti-GTP-Rac1 (#26903, NewEast Bioscience, PA) antibodies at 4 °C overnight.

Techniques: Control, Virus, Labeling, Activity Assay, Two Tailed Test, MANN-WHITNEY

Journal: PLoS Biology

Article Title: Neuregulin and BDNF Induce a Switch to NMDA Receptor-Dependent Myelination by Oligodendrocytes

doi: 10.1371/journal.pbio.1001743

Figure Lengend Snippet: (A) Western blots of control and NRG-treated cocultures for NR1, NR2B and its phosphorylated form (pNR2B), NR2C and its phosphorylated form (pNR2C), NR3A and NR3B, as well as for NR2A and NR2D compared to their respective positive controls of rat cortex (Ctx) and thalamus (Th); β-actin acts as a loading control throughout. (B) Densitometric quantification of subunit protein levels in cocultures (normalized to β-actin) in NRG normalized to the levels in control (NR2A and NR2D levels were undetectable). (C) Western blot of control (Con) and NRG-treated pure DRG cultures for NR3A and (below) densitometric quantification of subunit protein levels (normalized to β-actin and then to control). (D) Western blot for NR1 and NR3A of control (Con) and NRG-treated (for 6 d) pure OPC cultures, treated (+) or not treated (−) with 20 min glutamate (Glu, 100 µM) stimulation every day, with densitometric quantification of subunit protein levels (normalized to β-actin and then to control). The p values over the bars, in (B) from Holm–Bonferroni corrected t tests and in (C and D) from one-sample Student t tests, compare with control; numbers of experiments shown on bars.

Article Snippet: Immunoblots were then incubated overnight at 4°C with goat anti-Akt (Santa Cruz, 1∶1,000), rabbit anti-phosphorylated-Akt Ser473 (Cell Signalling, 1∶1,000), rabbit anti-phosphorylated-ERK1/2 Thr202/Tyr214 (Cell Signalling, 1∶1,000), mouse anti-ERK1/2 (Cell Signalling, 1∶2,000), rabbit anti-MBP (Sigma, 1∶3,000), mouse anti-NR1 (Millipore, 1∶1,000), rabbit anti-NR2A (Millipore, 1∶1,000), rabbit anti-NR2B (Abcam; 1∶1,000), rabbit anti-phosphorylated NR2B Tyr1472 (Millipore, 1∶1,000), rabbit anti-NR2C (Millipore, 1∶1,000), rabbit anti-phosphorylated NR2C S1096 (a kind gift from Katherine W. Roche, NINDS), rabbit anti-NR3A (Millipore, 1∶500), rabbit anti-NR3B (Millipore, 1∶300), rabbit anti-NR2D (Abcam, 1∶300), or mouse anti-β-actin (Sigma, 1∶100,000) in 3% BSA in PBS-T.

Techniques: Western Blot

Journal: Journal of Cardiovascular Pharmacology

Article Title: Cardiac N-methyl d-aspartate Receptors as a Pharmacological Target

doi: 10.1097/fjc.0000000000000424

Figure Lengend Snippet: FIGURE 7. Confirmation of the presence of GluN1, GluN2A, GluN2B, and GluN2C proteins in rat heart. Rat brain tissue homogenate was used as a control. Panel A shows the repre- sentative images in which the subunits were detected by immunoblotting. B, Changes in the GluN2B protein abun- dance in the heart with age of the animal. Actin was used as a loading control.

Article Snippet: Staining was performed with the following primary antibodies: mouse monolconal anti-GluN1 antibody, and rabbit polyclonal anti-GluN2C antibodies (NB300-118 and NB300-107; Novus Biologicals), and rabbit polyclonal anti-GluN2A and mouse monoclonal anti-GluN2B antibodies (ab14596 and ab28373; Abcam).

Techniques: Control, Western Blot