mouse anti‐glun2b  (NeuroMab)

Journal: Journal of Neurochemistry

Article Title: Regulation of Dendrite and Dendritic Spine Formation by TCF20

doi: 10.1111/jnc.16297

Figure Lengend Snippet: TCF20 knockdown alters neuronal proteins expression in total lysate and in synaptosomal fraction. (A) Representative Western blot and relative quantification of total lysate from shTCF20 or SCR transduced cortical neurons. Data are expressed as the mean ± SEM (2–3 mixed 6‐well plates obtained from n = 12 independent cultures; one‐sample t test; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). Statistical analysis: TCF20 degrees of freedom 26, t value 31.66, p value 2.71e‐22; GABRA1 degrees of freedom 16, t value 3.09, p value 0.0070; c‐FOS degrees of freedom 34, t value 2.08, p value 0.0454; GluN2B degrees of freedom 50, t value −0.105, p value 0.917; GluA2 degrees of freedom 20, t value −0.64, p value 0.530; GABRA5 degrees of freedom 16, t value −0.46, p value 0.650; GAD65 degrees of freedom 24, t value −0.072, p value 0.943; BDNF degrees of freedom 18, t value 4.72, p value 0.00017; PSD‐95° of freedom 44, t value 4.50, p value 4.88e‐05. (B) Representative Western blot and relative quantification of synaptosomal preparation from shTCF20 or SCR transduced cortical neurons. Data are expressed as the mean ± SEM (2–3 mixed 6‐well plates obtained from n = 9 independent cultures; one‐sample t test; ** p < 0.01; *** p < 0.001; **** p < 0.0001). Statistical analysis: GluN2B degrees of freedom 40, t value 4.72, p value 0.00000168; GluA2 degrees of freedom 14, t value −2.44, p value 0.0284; GAD65 degrees of freedom 10, t value 0.55, p value 0.5945; BDNF degrees of freedom 28, t value 4.41, p value 0.00014; PSD‐95° of freedom 14, t value 6.53, p value 0.0000134; GABRA1 degrees of freedom 10, t value 5.90, p value 0.00015.

Article Snippet: The following primary antibodies were used for western blot: rabbit anti‐β‐Actin (A206, 1:1000, Sigma, RRID:AB_476693), rabbit anti‐BDNF (PA5‐85730, 1:500, Invitrogen, RRID:AB_2792869), rabbit anti‐c‐Fos (9F6, 1:250, Cell Signaling, RRID:AB_2247211), mouse anti‐GABRA1 (75‐136, 1:1000 NeuroMab, RRID:AB_2877288), mouse anti‐GABRA5 (N415/24, 1:500, NeuroMab, RRID:AB_2877618), mouse anti‐GAD65 (D5G2/5843S, 1:1000, Cell Signaling, RRID:AB_10835855), mouse anti‐GluA2 (75‐002, 1:400, NeuroMab, RRID:AB_2232661), mouse anti‐GluN2B (75‐097, 1:400, Neuromab, RRID:AB_10673405), rabbit anti‐PSD‐95 (D27E11, 1:1000, Cell Signaling, RRID:AB_2943447), rabbit anti‐TCF20 (1:500 kindly provided by Dr. Sjøttem and published in Rekdal, Sjøttem, and Johansen ).

Techniques: Knockdown, Expressing, Western Blot

Journal: Journal of Neurochemistry

Article Title: Regulation of Dendrite and Dendritic Spine Formation by TCF20

doi: 10.1111/jnc.16297

Figure Lengend Snippet: RNA‐sequencing analyses. (A, B) Heatmap of the identified DEGs (hierarchical clustering, Pearson correlation, average function linkage and 4 as cut‐off z‐score) where a clear clustering of the control samples (SCR) over the shTCF20 is visible. (C) Principal component analysis shows a simple separation by the first principal component which explains 25% of the total variance between the SCR and shTCF20 group. (D) Differential gene expression analysis (DESeq2 method) reveals 116 downregulated and 213 upregulated genes in the SCR versus shTCF20 contrast. (E) Volcano plot evidencing relevant down‐expressing genes like Dlg4, BDNF, GABARAP, GRIN2b, and Gabaralp1.

Article Snippet: The following primary antibodies were used for western blot: rabbit anti‐β‐Actin (A206, 1:1000, Sigma, RRID:AB_476693), rabbit anti‐BDNF (PA5‐85730, 1:500, Invitrogen, RRID:AB_2792869), rabbit anti‐c‐Fos (9F6, 1:250, Cell Signaling, RRID:AB_2247211), mouse anti‐GABRA1 (75‐136, 1:1000 NeuroMab, RRID:AB_2877288), mouse anti‐GABRA5 (N415/24, 1:500, NeuroMab, RRID:AB_2877618), mouse anti‐GAD65 (D5G2/5843S, 1:1000, Cell Signaling, RRID:AB_10835855), mouse anti‐GluA2 (75‐002, 1:400, NeuroMab, RRID:AB_2232661), mouse anti‐GluN2B (75‐097, 1:400, Neuromab, RRID:AB_10673405), rabbit anti‐PSD‐95 (D27E11, 1:1000, Cell Signaling, RRID:AB_2943447), rabbit anti‐TCF20 (1:500 kindly provided by Dr. Sjøttem and published in Rekdal, Sjøttem, and Johansen ).

Techniques: RNA Sequencing Assay, Control, Expressing

Journal: Science Advances

Article Title: Synaptic rearrangement of NMDA receptors controls memory engram formation and malleability in the cortex

doi: 10.1126/sciadv.ado1148

Figure Lengend Snippet: ( A ) Experimental design (top) and time course of memory formation (bottom). Performance of EXP rats was robust and long-lasting with forgetting occurring only after 60 days (group × delay interaction: F 2,50 = 3.52; P < 0.05); * P < 0.05. ( B ) Immunoblots (top) and quantitative analysis (bottom) of NMDAR subunit expression in PSD-enriched membrane fractions from the OFC of FP and EXP rats. A progressive increase in the GluN2A/GluN2B ratio occurred as memory consolidated in the OFC of EXP rats (day 15, long-term memory; day 30, remote memory). At day 60, memory forgetting was associated with an increased contribution of GluN2B-containing NMDARs (group × delay interaction: F 3,40 = 7.20; P < 0.001); * P < 0.05. ( C ) Experimental design investigating memory persistence. EXP rats interacted socially with a demonstrator fed with cumin-flavored chow. Odor control (OC) rats were only able to smell cumin from a jar. ( D ) Enhanced preference for cumin-flavored food remained stable over 30 days in EXP rats but faded over the same time period in OC rats (group × delay interaction: F 2,45 = 4.44; P < 0.05). Performance of OC rats at day 30 was similar to that of FP controls, which interacted with a demonstrator fed with plain food. ( E ) Quantitative analysis of NMDAR subunit expression in PSD-enriched membrane fractions from the OFC of FP and OC rats tested at various intervals (days 1, 15, and 30) following social interaction. No redistribution of synaptic GluN2A and GluN2B subunits occurred over time in OC rats (group × delay interaction: F 2,43 = 0.013; P > 0.98, NS). * P < 0.05, **** P < 0.0001. (A), (B), (D), and (E), n = 4 to 11 rats per group.

Article Snippet: The blots were then incubated overnight at 4°C with a mouse anti-GluN2B antibody (RRID: AB_827426; catalog number: MAB5778; 1:1000; Millipore, Molsheim, France) and a rabbit anti-GluN2A antibody (RRID:AB_1163481; catalog number: 04-901; 1:1000; Millipore, Molsheim, France).

Techniques: Western Blot, Expressing, Membrane, Control

Journal: Science Advances

Article Title: Synaptic rearrangement of NMDA receptors controls memory engram formation and malleability in the cortex

doi: 10.1126/sciadv.ado1148

Figure Lengend Snippet: ( A ) Experimental design. Rats were injected chronically into the OFC with aCSF (vehicle) or NMDAR antagonists (AP5, TCN-201, or Ifenprodil) during the early (from day 1 to day 11) or late (from day 16 to day 26) post-acquisition periods and tested at day 30 following social interaction. ( B ) Early, but not late, post-encoding blockade of GluN2B subunits by intra-OFC infusions of ifenprodil impaired remote memory retrieval at day 30. The opposite pattern was observed when antagonizing GluN2A-NMDARs with TCN-201. Both early and late OFC infusions of the nonselective NMDAR antagonist AP5 impaired remote memory retrieval (early treatment effect: F 3,32 = 6.73, P < 0.05; late treatment effect: F 3,37 = 4.09, P < 0.05). * P < 0.05 versus aCSF (vehicle control), n = 8 to 13 rats per group. ( C ) Experimental design investigating relearning. Animals infused with aCSF (vehicle) or AP5 into the OFC during the early (from day 1 to day 11) or late (from day 16 to day 26) post-acquisition periods were submitted to a second interaction with a different flavor (cocoa) 1 week later (day 37). This novel memory for cocoa was assessed 30 days later to enable the establishment of remote memory in the OFC (day 67, choice between cocoa and cinnamon). ( D ) Groups previously injected with aCSF or AP5 during the early or the late post-acquisition periods exhibited a similar acquired preference for cocoa (group × delay: F 1,15 = 0.51, P > 0.48, NS, n = 8 to 13 rats per group). The dotted line in (B) and (D) represents innate preference of FP control rats.

Article Snippet: The blots were then incubated overnight at 4°C with a mouse anti-GluN2B antibody (RRID: AB_827426; catalog number: MAB5778; 1:1000; Millipore, Molsheim, France) and a rabbit anti-GluN2A antibody (RRID:AB_1163481; catalog number: 04-901; 1:1000; Millipore, Molsheim, France).

Techniques: Injection, Control

Journal: Science Advances

Article Title: Synaptic rearrangement of NMDA receptors controls memory engram formation and malleability in the cortex

doi: 10.1126/sciadv.ado1148

Figure Lengend Snippet: ( A and B ) Early, but not late, hippocampal inactivation with 6-cyano-7-nitroquinoxaline2,3-dione (CNQX) prevented the consolidation-induced rearrangement of cortical GluN2A and GluN2B subunit–containing NMDARs in the OFC ( F 1,16 = 9.86; P < 0.001); * P < 0.05, n = 5 rats per group. ( C to E ), Memory strength (one versus four social interactions) modulated the post-acquisition kinetics of hippocampal and cortical involvement upon memory retrieval at day 7. CNQX-induced disruption of hippocampal activity after one interaction was no longer observed after four interactions (D, treatment × interaction number: F 2,37 = 4.519; P < 0.05); * P < 0.05, n = 6 to 9 rats per group). OFC dependency manifested after four, but not one, interactions (E, F 2,39 = 12.60; P < 0.0001); ** P < 0.01, n = 7 to 9 rats per group). ( F ) Immunoblots (top) and corresponding quantitative analysis (bottom). An increase in the GluN2A-/GluN2B-NMDAR ratio was observed in the OFC at day 7 after four, but not one, interactions ( F 2,13 = 16.51; P = 0.0003); ** P < 0.01, *** P < 0.001, n = 5 to 7 rats per group. ( G and H ) Accordingly, blocking GluN2A-containing NMDARs with TCN-201 during the early post-encoding phase impaired remote memory retrieval at day 30 while being ineffective in the single interaction condition ( F 1,26 = 17.22; P = 0.0003); * P < 0.05, ** P < 0.01, n = 8 rats per group; data from are shown for comparison).

Article Snippet: The blots were then incubated overnight at 4°C with a mouse anti-GluN2B antibody (RRID: AB_827426; catalog number: MAB5778; 1:1000; Millipore, Molsheim, France) and a rabbit anti-GluN2A antibody (RRID:AB_1163481; catalog number: 04-901; 1:1000; Millipore, Molsheim, France).

Techniques: Disruption, Activity Assay, Western Blot, Blocking Assay, Comparison

Journal: Science Advances

Article Title: Synaptic rearrangement of NMDA receptors controls memory engram formation and malleability in the cortex

doi: 10.1126/sciadv.ado1148

Figure Lengend Snippet: ( A ) Experimental design investigating memory specificity. ( B ) Intra-OFC infusions of the GluN2B-NMDAR antagonist ifenprodil at the time of the second interaction (cumin) impaired remote memory for this second flavor while sparing remote memory for the first one (cocoa) (treatment × flavor pair: F 1,36 = 11.90; P = 0.0014). The nonselective NMDAR antagonist AP5 exhibited a similar profile ( F 1,32 = 17.31; P = 0.0002). The GluN2A-NMDAR antagonist TCN-201 was ineffective regardless of the flavor pair (main treatment effect: F 1,34 = 0.28; P = 0.59, NS). ( C ) Similar effects of AP5 infusions into the OFC were obtained when the two cumin/thyme and cocoa/cinnamon flavor pairs were counterbalanced (flavor × treatment: F 1,24 = 16.36, P < 0.001). ( D ) Experimental design investigating encoding processes. ( E ) Intra-OFC infusions of AP5, TCN-201, and ifenprodil before social interaction did not impair performance of rats tested 4 days (D4) later (treatment effect: F 3,26 = 1.207, P > 0.33, NS). ( F ) Experimental design investigating CaMKII contribution. ( G ) Intra-OFC infusions of the CaMKII inhibitor CN21 impaired formation of a newly encoded memory without destabilizing a previously encoded information undergoing consolidation ( F 2,49 = 23.92; P < 0.0001). ( H ) (Top) Efficacy of intra-OFC infusions of GluN2A- and GluN2B-NMDAR antagonists to impair remote memory retrieval at day 30 as a function of the specific phases of systems consolidation. Graph is based on findings from and (B). (Bottom) Schematic of the GluN2A- and GluN2B-NMDAR redistributions at cortical synapses as enduring memories mature in the cortex. Size variation of NMDAR pictograms represents changes in the functional contribution of GluN2A- (in brown) and GluN2B-NMDARs (in pink). (B), (C), (E), and (G), n = 6 to 12 rats per group; * P < 0.05, **** P < 0.0001.

Article Snippet: The blots were then incubated overnight at 4°C with a mouse anti-GluN2B antibody (RRID: AB_827426; catalog number: MAB5778; 1:1000; Millipore, Molsheim, France) and a rabbit anti-GluN2A antibody (RRID:AB_1163481; catalog number: 04-901; 1:1000; Millipore, Molsheim, France).

Techniques: Functional Assay

Journal: Science Advances

Article Title: Synaptic rearrangement of NMDA receptors controls memory engram formation and malleability in the cortex

doi: 10.1126/sciadv.ado1148

Figure Lengend Snippet: ( A to D ) Intra-OFC infusion of an anti-GluN2B, but not of an anti-GluN2A, antibody during the early post-encoding period (A) impaired remote memory retrieval assessed 30 days later (B, group × treatment interaction: F 1,38 = 16.10; P = 0.0003) and abolished the increase in GluN2A-/GluN2B-NMDAR ratio observed in the OFC of control rats (D, F 2,19 = 10.45; P = 0.0009); * P < 0.05, ** P < 0.01, n = 8 to 9 rats per group. ( E to H ) Intra-OFC delivery of the anti-GluN2B antibody during the late post-encoding period (E) prevented forgetting (F, F 2,32 = 7.67; P = 0.0019) and the decrease of the GluN2A-/GluN2B-NMDAR ratio in the OFC (H, F 2,18 = 5.88; P = 0.0109) normally seen in control rats at day 60. * P < 0.05, # P < 0.05, n = 10 to 14 rats per group. Immunoblots in (C) and (G) served for quantification of respective GluN2A-/GluN2B-NMDAR ratios.

Article Snippet: The blots were then incubated overnight at 4°C with a mouse anti-GluN2B antibody (RRID: AB_827426; catalog number: MAB5778; 1:1000; Millipore, Molsheim, France) and a rabbit anti-GluN2A antibody (RRID:AB_1163481; catalog number: 04-901; 1:1000; Millipore, Molsheim, France).

Techniques: Control, Western Blot

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: GluN2B are necessary for cocaine-induced silent synapses and addiction memory. (A) Schematic for shGluN2B or control virus injection in the NAcSh (left). A timeline for general experiments-cocaine or saline injections were made once every day (5 days, top, right) and expression of shGluN2B virus in NAcSh area (bottom, right). Scale bar = 100 μm. (B) Representative images of GluN2B-positive puncta (red) in D1-MSNs dendrites (green, left). GluN2B puncta density after cocaine administration is compared between mice that received either shGluN2B or control virus (n = 13 cells for cKD; n = 14 cells for control, right). Scale bar = 5 μm. (C) Immuno-electron microscopic images showing subcellular localization of GluN2B (18 nm gold particles, blue arrows) and eYFP for labeling D1-MSNs (6 nm gold particles, green arrows, left). GluN2B particle density is compared between cocaine/cKD and cocaine/control D1-MSNs (n = 9 cells for cKD; n = 8 cells for control, right). Scale bars = 100 nm. ( D) Schematic for optogenetic stimulation of the BLA-NAcSh pathway (left). Representative EPSC traces from optical stimulation (blue bar) of BLA glutamatergic inputs with a picture with (red) or without NBQX (black) showing cKD D1-MSNs (an inserted image, right). ( E) Sample traces of success (black trace) and failure (red trace) responses by the optical minimal stimulation for AMPAR-EPSCs (−70mV) and NMDAR-EPSCs (+50mV, left). Example plots of optical responses with minimal optical stimulation in designated groups (failure trials in red dots, successful trials in black dots, middle). The proportion of silent synapses is compared between cocaine-treated mice that previously received either shGluN2B or control virus (n = 20 cells for cKD; n = 23 cells for control group, right). (F) Schematic for conditional place preference (left). Heatmap traces of representative animals (middle). Quantified CPP scores to the cocaine chamber are compared between mice that previously received either shGluN2B or control virus (n = 10 mice for cKD; n = 17 mice for control group, right). (G) Schematic for cKO of GluN2B using CRISPR-Cas9 (left). Representative images for GluN2B (cyan) expression in D1-MSNs expressing Cas9-eGFP (green) and sgGrin2b-mCherry (red, middle). GluN2B puncta density after cocaine administration is compared between mice that previously received either sgGrin2b or control virus (n = 19 cells for cKO; n = 29 cells for control group, right). Scale bar = 20 μm. (H) Immuno-electron microscopic images showing subcellular localization of GluN2B (12 nm gold particles, blue arrows), mCherry (6 nm gold particles, red arrows) and eYFP for labeling D1-MSNs (18 nm gold particles, green arrows, left). GluN2B particle density is compared in the dendrites-labelled by eYFP between cocaine/cKO and cocaine/control group (n = 20 cells for cKO; n = 18 cells for control group, right). Scale bar = 100 nm. (I) Example plots of AMPAR- and NMDAR-EPSCs with minimal optical stimulation in designated groups (failure trials in red dots; successful trials in black dots, left). The proportion of silent synapses is compared between cocaine-treated Cas9-eGFP mice that previously received either sgGrin2b or control virus (n = 17 cells for cKO; n = 13 cells for the control group, right). ( J) Quantified CPP scores in the cocaine chamber are compared between GluN2B cKO and control groups (n = 17 mice for cKO; n = 13 mice for the control group). Data are represented as mean ± SEM (error bars); **p < 0.01, ***p < 0.001, ****p < 0.0001 by two-tailed unpaired t test or Mann-Whitney test.

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: Virus, Injection, Saline, Expressing, Labeling, CRISPR, Two Tailed Test, MANN-WHITNEY

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: Structural and functional maturation of synapses are accelerated by GluN2B depletion. (A) A chematic for the 3-dimensional reconstruction from serial SEM images (left). Representative illustrations of reconstructed dendrites from cocaine-treated cKD and control D1-MSNs (right). (B) The proportion of morphologically-categorized spine classes between cocaine-treated mice that previously received either shGluN2B (cocaine/cKD, n = 79 spines from 3 mice) or control virus (cocaine/control, n = 85 spines mice from 2 mice). ( C) Analysis of averaged spine head widths, PSD areas, and density of branched spines are compared between cocaine/cKD and cocaine/control groups. (head widths and PSD areas, n = 85 units for cocaine/control, n = 95 units for cocaine/cKD; density of branched spines, n = 4 units for cocaine/control, n = 4 units or cocaine/cKD). ( D) Sample traces of mEPSCs from D1-MSNs of cocaine-treated mice that previously received either shGluN2B or control virus. ( E) A cumulative probability plot of mEPSC inter-event intervals (IEIs) and an inserted summary histogram of quantified mEPSC frequency after cocaine exposure (n = 25 cells each for control and cKD groups, left). A cumulative probability plot of mEPSC amplitudes and a summary histogram for quantified mEPSC amplitudes after cocaine exposure (n = 25 cells each for cocaine/control and cocaine/cKD groups, right). ( F) Sample traces of optically-evoked EPSCs with stimulation of 50-ms interval in D1-MSNs of saline-treated mice that previously received either shGluN2B or control virus (left). Measurement of paired-pulse ratios (PPRs) obtained with stimulation of 50-, 100-, and 150-ms intervals in D1-MSNs from cocaine-treated mice that previously received either shGluN2B or control virus (n = 9 cells for cKD; n = 11 cells for control groups, right). (G) Sample traces of AMPAR-(at −70 mV) and NMDAR-(+50 mV) EPSCs in cocaine-treated D1-MSNs (left). A/N ratios are compared between cocaine-treated mice that previously received either shGluN2B or control virus (n = 22 cells for cKD; n = 15 cells for control groups, right). Data are represented as mean ± SEM (error bars); *p < 0.05, **p < 0.01, ****p < 0.0001 by two-tailed unpaired t test or Mann-Whitney test.

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: Functional Assay, Virus, Saline, Two Tailed Test, MANN-WHITNEY

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: GluN2B ablation facilitated the recruitment of GluA2-AMPARs. ( A) A variance-current plot from cocaine-treated D1-MSNs (red for cKD; black for control groups, left). Averaged single-channel currents are compared between cocaine-treated mice that previously received either shGluN2B or control virus (n = 21 cells for cKD; n = 13 cells for control groups, right). ( B) Sample traces of evoked EPSCs before (black) and after (magenta) treatment with NASPM, an antagonist for GluA2-lacking AMPARs in D1-MSNs from cocaine-treated mice that previously received either shGluN2B or control virus (left). The sensitivity to NASPM is compared between cocaine/cKD (n = 13 cells) and cocaine/control (n = 15 cells) groups (right). ( C) Representative images of GluA2-positive puncta (red) in cocaine-treated D1-MSNs dendrites (green, left). Quantification of GluA2 puncta on D1-MSNs dendrites between cocaine-treated mice that previously received either shGluN2B or control virus (n = 9 cells for cKD; n = 11 cells fo control groups, right). Scale bar = 5 µm. ( D) Immuno-electron microscopic images for subcellular localization of GluA2 (6 nm gold particles, magenta arrows) and eYFP for labeling of D1-MSNs (18 nm gold particles, green arrows, left). GluA2 particles present within the synaptic areas are compared between cocaine-treated mice that received either shGluN2B or control virus (n = 7 cells for cKD; n = 10 cells for control groups, right). Scale bars = 100 nm. ( E) An experimental timeline for measurement of cocaine-induced locomotor activity. Locomotor activity was monitored upon cocaine infusion on a daily basis between groups (left). Cocaine-induced locomotion is compared between cKD (red, n =16 mice) and control (black, n = 14 mice) groups (right). ( F) Cocaine-induced locomotion is compared between cKO (orange, n = 8 mice) and control (black, n = 5 mice) groups. Data are represented as mean ± SEM (error bars); *p < 0.05, **p < 0.01, ***p < 0.001 by two-tailed unpaired t test, Mann-Whitney 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: Virus, Labeling, Activity Assay, Two Tailed Test, MANN-WHITNEY

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: GluN2B depletion induced impairment in addiction memory and elevation of behavioral sensitization attributed to GluA2-AMPAR trafficking . (A) A schematic for Pep2m treatment (left). Representative images showing the tips of guide cannulas (dashed line) in the NAcSh (right). Scale bars = 500 μm and 100 μm. ( B) Representative images for GluA2-positive puncta (red) in dendrites of D1-MSNs (green) from cocaine/cKD mice (left). Scale bar = 10 µm. GluA2 particle density in eYFG-labelled dendrites is compared between GluN2B cKD mice where either Pep2m or vehicle was infused into NAcSh areas (n = 13 cells for Pep2m; n = 15 cells for vehicle groups, right). ( C) An experimental timeline for behavioral measurement after Pep2m or vehicle infusion. ( D) Quantified CPP scores in the cocaine chamber after Pep2m or vehicle treatment are compared in mice that previously received shGluN2B virus (n = 8 mice for Pep2m; n = 3 mice for vehicle groups). ( E) Locomotor activity of GluN2B cKD mice is compared upon daily cocaine infusion after Pep2m or vehicle treatment (n = 5 for Pep2m; n = 3 for vehicle groups). Data are represented as mean ± SEM (error bars); *p < 0.05, ****p < 0.0001 by two-tailed unpaired test, Mann-Whitney 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: Virus, Activity Assay, Two Tailed Test, MANN-WHITNEY

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: Rac1-controls the trafficking of GluA2-AMPARs. (A) Representative confocal images of active Rac1-positive puncta (GTP-Rac1, red) in cocaine-treated D1-MSNs (green, left). Scale bar = 5 μm. Density of GTP-Rac1-positive puncta after cocaine administration is compared between mice that previously received either shGluN2B or control virus (n = 11 cells for cKD; n = 11 cells for control groups, right). ( B) Experimental schematic for photoillumination-triggered modulation of Rac1 activity (left). GTP-Rac1 puncta density after photoillumination is compared between cocaine/cKD mice that previously received PA-Racl or PI-Rac1 virus (n = 5 cells for PA-Rac1; n = 7 cells for PI-Rac1, right). ( C) Representative confocal images of active GluA2-positive puncta (red) on the dendrites of cocaine/cKD D1-MSNs (green) expressing PA-or PI-Rac1 (left). Scale bar = 5 μm. Quantified immuno-labeled puncta of GluA2 after photoillumination is compared between cocaine/cKD mice that previously received either PA-Rac1 or PI-Rac1 virus (n = 8 cells for PA-Rac1; n = 14 cells for PI-Rac1 groups, right). ( D) Sample traces of AMPAR- and NMDAR-EPSCs in cocaine/cKD D1-MSNs after photoillumination of PI or PA-Rac1 (left). A/N ratios are compared after photoillumination between cocaine/cKD mice that received either PA-Rac1 or PI-Rac1 virus (n = 10 cells for PA-Rac1; n = 19 cells for PI-Rac1 groups, right). ( E) Quantified CPP scores to the cocaine-paired chamber are compared after photoillumination between cKD mice that received either PA-Rac1 or PI-Rac1 virus (n = 9 mice for PA-Rac1; n = 10 mice for PI-Rac1 groups). ( F) Left: a photoillumination timeline for measurement of locomotor activity. Light was delivered during and after cocaine infusion. Right: cocaine-induced locomotor activity is compared in GluN2B cKD animals that received either PA-Rac1 or PI-Rac1 virus (n = 3 mice for PA-Rac1; n = 4 mice for PI-Rac1 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, 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: Virus, Activity Assay, Expressing, Labeling, Two Tailed Test, MANN-WHITNEY

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