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: After blocking, membranes were incubated overnight at 4°C with primary antibodies, mouse anti-GluNR1 (1:1,000 for #05-432, Millipore, MA), rabbit anti-GluNR2A (1:1,000 for #07-632, Millipore, MA), rabbit anti-GluNR2B (1:1,000 for #AGC-003, Alomone lab) or mouse anti-GluN2B (1:500 for #610416, BD Transduction Lab, NJ), rabbit anti-NR2C (1:1,000 for #AGC-018, Alomone lab), rabbit anti-GluA1(1:2,000 for #13185, Cell signaling Technology, MA), mouse anti-GluA2 (1:2,000 for #MAB397, Milipore) and anti-β-actin (1: 10,000 for #A5441, Sigma).
Techniques: Control, Virus, Labeling, Activity Assay, Two Tailed Test, MANN-WHITNEY
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