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Cholesterol depletion reduces synaptic localization of NMDARs. ( A , C ) Colocalization of surface GluN2A (A, green) or <t>GluN2B</t> (C, green) and the postsynaptic marker Shank (red) in control and cholesterol-depleted neurons (10 mM MβCD pretreatment, 5 min). Scale bar 2 µm. ( B , D ) Bar graphs showing Pearson's coefficient for the colocalization indicate the reduction of synaptic localization of GluN2A and GluN2B after cholesterol depletion. ( E ) Colocalization of surface GluA1 (green) and the postsynaptic marker Shank (red) in control and cholesterol-depleted neurons (MβCD). Scale bar 2 µm. ( F ) Bar graph showing Pearson's coefficient for the colocalization. ( G ) Examples of typical dual AMPAR-NMDAR mEPSCs in control autaptic neurons having various AMPAR to NMDAR ratio. ( H ) Examples of typical dual AMPAR-NMDAR mEPSCs in 10 mM MβCD-pretreated autaptic neurons. ( I ) Examples of NMDAR mEPSCs obtained from average dual mEPSCs after AMPAR mEPSC subtraction. A control neuron (top trace) and a cholesterol-depleted neuron (bottom trace). The arrows indicate mEPSC onsets. ( J ) The comparison of average amplitude of NMDAR mEPSCs in control neurons and in cholesterol-depleted neurons. (* p

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1) Product Images from "Cholesterol modulates presynaptic and postsynaptic properties of excitatory synaptic transmission"

Article Title: Cholesterol modulates presynaptic and postsynaptic properties of excitatory synaptic transmission

Journal: Scientific Reports

doi: 10.1038/s41598-020-69454-5

Cholesterol depletion reduces synaptic localization of NMDARs. ( A , C ) Colocalization of surface GluN2A (A, green) or GluN2B (C, green) and the postsynaptic marker Shank (red) in control and cholesterol-depleted neurons (10 mM MβCD pretreatment, 5 min). Scale bar 2 µm. ( B , D ) Bar graphs showing Pearson's coefficient for the colocalization indicate the reduction of synaptic localization of GluN2A and GluN2B after cholesterol depletion. ( E ) Colocalization of surface GluA1 (green) and the postsynaptic marker Shank (red) in control and cholesterol-depleted neurons (MβCD). Scale bar 2 µm. ( F ) Bar graph showing Pearson's coefficient for the colocalization. ( G ) Examples of typical dual AMPAR-NMDAR mEPSCs in control autaptic neurons having various AMPAR to NMDAR ratio. ( H ) Examples of typical dual AMPAR-NMDAR mEPSCs in 10 mM MβCD-pretreated autaptic neurons. ( I ) Examples of NMDAR mEPSCs obtained from average dual mEPSCs after AMPAR mEPSC subtraction. A control neuron (top trace) and a cholesterol-depleted neuron (bottom trace). The arrows indicate mEPSC onsets. ( J ) The comparison of average amplitude of NMDAR mEPSCs in control neurons and in cholesterol-depleted neurons. (* p

Figure Legend Snippet: Cholesterol depletion reduces synaptic localization of NMDARs. ( A , C ) Colocalization of surface GluN2A (A, green) or GluN2B (C, green) and the postsynaptic marker Shank (red) in control and cholesterol-depleted neurons (10 mM MβCD pretreatment, 5 min). Scale bar 2 µm. ( B , D ) Bar graphs showing Pearson's coefficient for the colocalization indicate the reduction of synaptic localization of GluN2A and GluN2B after cholesterol depletion. ( E ) Colocalization of surface GluA1 (green) and the postsynaptic marker Shank (red) in control and cholesterol-depleted neurons (MβCD). Scale bar 2 µm. ( F ) Bar graph showing Pearson's coefficient for the colocalization. ( G ) Examples of typical dual AMPAR-NMDAR mEPSCs in control autaptic neurons having various AMPAR to NMDAR ratio. ( H ) Examples of typical dual AMPAR-NMDAR mEPSCs in 10 mM MβCD-pretreated autaptic neurons. ( I ) Examples of NMDAR mEPSCs obtained from average dual mEPSCs after AMPAR mEPSC subtraction. A control neuron (top trace) and a cholesterol-depleted neuron (bottom trace). The arrows indicate mEPSC onsets. ( J ) The comparison of average amplitude of NMDAR mEPSCs in control neurons and in cholesterol-depleted neurons. (* p

Techniques Used: Marker

$Cholesterol depletion reduces the fraction of synaptic immobile NMDARs. ( A ) Surface NMDARs were detected using a QD-antibody complex directed against extracellular epitopes in GluN2A or GluN2B. Left, representative summed trajectories of NMDAR-QDs (red) acquired over a period of 25 s (20 Hz frame rate) in hippocampal neurons. Scale bar 5 µm. Right, representative examples of NMDAR reconstructed trajectories. ( B , C ) Diffusion coefficients for synaptic GluN2A-containing NMDARs and GluN2B-containing NMDARs in control and after cholesterol depletion (10 mM MβCD pretreatment, 5 min). ( D , E ) Diffusion coefficients for extrasynaptic GluN2A-containing NMDAR and GluN2B-containing NMDARs in control and after cholesterol depletion. ( F , G ) Diffusion coefficients for the mobile fraction of synaptic GluN2A and GluN2B-containing NMDARs in control and after cholesterol depletion. ( H , I ) Fraction of synaptic immobile receptors in control and after cholesterol depletion. (* p$
Figure Legend Snippet: Cholesterol depletion reduces the fraction of synaptic immobile NMDARs. ( A ) Surface NMDARs were detected using a QD-antibody complex directed against extracellular epitopes in GluN2A or GluN2B. Left, representative summed trajectories of NMDAR-QDs (red) acquired over a period of 25 s (20 Hz frame rate) in hippocampal neurons. Scale bar 5 µm. Right, representative examples of NMDAR reconstructed trajectories. ( B , C ) Diffusion coefficients for synaptic GluN2A-containing NMDARs and GluN2B-containing NMDARs in control and after cholesterol depletion (10 mM MβCD pretreatment, 5 min). ( D , E ) Diffusion coefficients for extrasynaptic GluN2A-containing NMDAR and GluN2B-containing NMDARs in control and after cholesterol depletion. ( F , G ) Diffusion coefficients for the mobile fraction of synaptic GluN2A and GluN2B-containing NMDARs in control and after cholesterol depletion. ( H , I ) Fraction of synaptic immobile receptors in control and after cholesterol depletion. (* p

Techniques Used: Diffusion-based Assay

2) Product Images from "Lack of the Actin Capping Protein, Eps8, Affects NMDA-Type Glutamate Receptor Function and Composition"

Article Title: Lack of the Actin Capping Protein, Eps8, Affects NMDA-Type Glutamate Receptor Function and Composition

Journal: Frontiers in Molecular Neuroscience

doi: 10.3389/fnmol.2018.00313

Increased GluN2B and reduced GluN2A synaptic content of NMDARs in Eps8 KO neurons. (A) Representative traces of NMDAR mediated mEPSCs recorded from WT and EPS8 KO hippocampal neurons in the presence or not of the GluN1/GluN2B blocker Ifenprodil (3 μM). Scale bar 10 pA, 50 ms. (B,C) Quantification of the inhibitory effect of ifenprodil on the NMDA-mEPSC frequency and amplitude respectively showing increased inhibition in KO neurons with respect to WT (B: Mann Whitney Test * P = 0.0270; C: Unpaired t test ** P = 0.0065). (D) Examples of whole-cell currents elicited in WT and EPS8 KO hippocampal neurons by application of a saturating concentration of NMDA (200 μm) in the presence or absence of GluN2B-selective antagonist ifenprodil (3 μM). Scale bar 300 pA, 2 s. Quantitation of current density (E) , %age of ifenprodil inhibition (F) showing a larger amount of ifenprodil-dependent inhibition in KO neurons with respect to WT (Mann Whitney Test * P = 0.0127). (G) Summary distribution graph of whole-cell currents elicited in WT and EPS8 KO treated or not with tricine (10 mM) showing a reduced GluN2A-dependent increase of current density upon tricine exposure in KO neurons with respect to WT (Mann Whitney Test, *** P

Figure Legend Snippet: Increased GluN2B and reduced GluN2A synaptic content of NMDARs in Eps8 KO neurons. (A) Representative traces of NMDAR mediated mEPSCs recorded from WT and EPS8 KO hippocampal neurons in the presence or not of the GluN1/GluN2B blocker Ifenprodil (3 μM). Scale bar 10 pA, 50 ms. (B,C) Quantification of the inhibitory effect of ifenprodil on the NMDA-mEPSC frequency and amplitude respectively showing increased inhibition in KO neurons with respect to WT (B: Mann Whitney Test * P = 0.0270; C: Unpaired t test ** P = 0.0065). (D) Examples of whole-cell currents elicited in WT and EPS8 KO hippocampal neurons by application of a saturating concentration of NMDA (200 μm) in the presence or absence of GluN2B-selective antagonist ifenprodil (3 μM). Scale bar 300 pA, 2 s. Quantitation of current density (E) , %age of ifenprodil inhibition (F) showing a larger amount of ifenprodil-dependent inhibition in KO neurons with respect to WT (Mann Whitney Test * P = 0.0127). (G) Summary distribution graph of whole-cell currents elicited in WT and EPS8 KO treated or not with tricine (10 mM) showing a reduced GluN2A-dependent increase of current density upon tricine exposure in KO neurons with respect to WT (Mann Whitney Test, *** P

Techniques Used: Mass Spectrometry, Inhibition, MANN-WHITNEY, Concentration Assay, Quantitation Assay

$Increased levels of GluN2B-containing and Y1472-GluN2B phosphorylated NMDARs in synaptic triton insoluble fraction (TIF) from Eps8 KO tissue. (A) Western blotting (WB) analysis confirmed that TIF preparation was actually enriched in postsynaptic proteins. (B–D) WB analysis and relative quantification in homogenate and TIF fractions for the indicated antibodies. All the WB were run twice (data are mean ± SEM, N = 4; Kruskal-Wallis test followed by Dunn’s multiple Comparison test * P$
Figure Legend Snippet: Increased levels of GluN2B-containing and Y1472-GluN2B phosphorylated NMDARs in synaptic triton insoluble fraction (TIF) from Eps8 KO tissue. (A) Western blotting (WB) analysis confirmed that TIF preparation was actually enriched in postsynaptic proteins. (B–D) WB analysis and relative quantification in homogenate and TIF fractions for the indicated antibodies. All the WB were run twice (data are mean ± SEM, N = 4; Kruskal-Wallis test followed by Dunn’s multiple Comparison test * P

Techniques Used: Western Blot

3) Product Images from "Surface dynamics of GluN2B-NMDA receptors controls plasticity of maturing glutamate synapses"

Article Title: Surface dynamics of GluN2B-NMDA receptors controls plasticity of maturing glutamate synapses

Journal: The EMBO Journal

doi: 10.1002/embj.201386356

Regulation of GluN2B-NMDAR surface dynamics by CAMKII and CKII activities Representative trajectories (25-s duration, 20-Hz acquisition) of surface QD-conjugated GluN2B-NMDAR within Homer 1c-GFP-labeled synaptic areas (gray) before and after chem LTP in immature hippocampal neuron (9–12 div) in control (green), TBB (orange), and KN62 (red) conditions. Scale bar = 1 μm. Synaptic GluN2B-NMDAR surface diffusion change (normalized to baseline for each condition) after chem LTP in immature neurons (

Figure Legend Snippet: Regulation of GluN2B-NMDAR surface dynamics by CAMKII and CKII activities Representative trajectories (25-s duration, 20-Hz acquisition) of surface QD-conjugated GluN2B-NMDAR within Homer 1c-GFP-labeled synaptic areas (gray) before and after chem LTP in immature hippocampal neuron (9–12 div) in control (green), TBB (orange), and KN62 (red) conditions. Scale bar = 1 μm. Synaptic GluN2B-NMDAR surface diffusion change (normalized to baseline for each condition) after chem LTP in immature neurons (

Techniques Used: Labeling, Diffusion-based Assay

Antibodies against extracellular epitopes of NMDAR from autoimmune encephalitis patients acutely prevent chem LTP Schematic diagram of the anti-NMDAR IgG isolation procedure from anti-NMDAR encephalitis patients. The cerebrospinal fluid (CSF) was collected and IgG were purified for in vitro imaging experiments. Lower panels: note the high co-localization of surface staining from surface patient anti-NMDAR IgG (“sPat. IgG,” green) and commercial anti-GluN1 antibodies (“sGluN1,” red). Scale bar = 1 μm. Representative GluN2B-NMDAR-QD trajectories from neurons incubated either with control or with patient IgG. Note the massive reduction in surface dynamics. Scale bar = 250 nm. Representative images of hippocampal neurons in the basal conditions or after glutamate (30 μM) application. The pseudocolor representation shows the different intensity levels of the calcium indicator (Fluo4-AM, 2 μM) before and after the glutamate stimulation. Neurons were incubated either with no IgG, controls' IgG (Cont. IgG), or patients' IgG (Pat. IgG). Scale bar = 20 μm. Right panel: Average calcium intensity change (ΔF/F0) over time after glutamate stimulation of hippocampal neurons in no IgG, controls' IgG (Cont. IgG), or patients' IgG (Pat. IgG) conditions. Hippocampal neurons expressing either GluN1-SEP or GluA1-SEP were incubated with IgG (5 μg/ml) either from control or from anti-NMDAR patients for 20–25 min. Note that patient IgG do not affect GluN1-SEP distribution. Neurons were stimulated with a chem LTP protocol and each synaptic GluA1-AMPAR cluster was followed over time. Note that chem LTP increased the intensity of GluA1-SEP in synaptic clusters (arrows) only in control IgG condition. Scale bars = 1 μm. Lower panels: Quantification of the GluA1-AMPAR synaptic content and percentage of potentiated GluA1-AMPAR synapses in control or patient IgG conditions. For each neuron, GluA1 synaptic fluorescence intensity was quantified before and 10–15 min after chem LTP. The GluA1-AMPAR synaptic content and percentage of potentiated GluA1-AMPAR synapses significantly increased in control condition ( n = 6 neurons; Student's t -test, * P

Figure Legend Snippet: Antibodies against extracellular epitopes of NMDAR from autoimmune encephalitis patients acutely prevent chem LTP Schematic diagram of the anti-NMDAR IgG isolation procedure from anti-NMDAR encephalitis patients. The cerebrospinal fluid (CSF) was collected and IgG were purified for in vitro imaging experiments. Lower panels: note the high co-localization of surface staining from surface patient anti-NMDAR IgG (“sPat. IgG,” green) and commercial anti-GluN1 antibodies (“sGluN1,” red). Scale bar = 1 μm. Representative GluN2B-NMDAR-QD trajectories from neurons incubated either with control or with patient IgG. Note the massive reduction in surface dynamics. Scale bar = 250 nm. Representative images of hippocampal neurons in the basal conditions or after glutamate (30 μM) application. The pseudocolor representation shows the different intensity levels of the calcium indicator (Fluo4-AM, 2 μM) before and after the glutamate stimulation. Neurons were incubated either with no IgG, controls' IgG (Cont. IgG), or patients' IgG (Pat. IgG). Scale bar = 20 μm. Right panel: Average calcium intensity change (ΔF/F0) over time after glutamate stimulation of hippocampal neurons in no IgG, controls' IgG (Cont. IgG), or patients' IgG (Pat. IgG) conditions. Hippocampal neurons expressing either GluN1-SEP or GluA1-SEP were incubated with IgG (5 μg/ml) either from control or from anti-NMDAR patients for 20–25 min. Note that patient IgG do not affect GluN1-SEP distribution. Neurons were stimulated with a chem LTP protocol and each synaptic GluA1-AMPAR cluster was followed over time. Note that chem LTP increased the intensity of GluA1-SEP in synaptic clusters (arrows) only in control IgG condition. Scale bars = 1 μm. Lower panels: Quantification of the GluA1-AMPAR synaptic content and percentage of potentiated GluA1-AMPAR synapses in control or patient IgG conditions. For each neuron, GluA1 synaptic fluorescence intensity was quantified before and 10–15 min after chem LTP. The GluA1-AMPAR synaptic content and percentage of potentiated GluA1-AMPAR synapses significantly increased in control condition ( n = 6 neurons; Student's t -test, * P

Techniques Used: Isolation, Purification, In Vitro, Imaging, Staining, Incubation, Expressing, Fluorescence

$The activity-dependent shift in CaMKII dynamics within dendritic spines is regulated by GluN2B-NMDAR dynamics Representative immunoblots showing the immunoprecipitation (IP) of CaMKII (α form) and phospho-CaMKII-Thr286 with GluN2B in membrane fractions from hippocampal slices (P17–20 rats) incubated with buffer (control) or GluN1 x-link. Lower panel: the ratio between CaMKII and GluN2B optical densities is represented ( n = 3 independent experiments). SM, start material; No Ab., no antibody; Cont., control. CaMKII-GFP was detected and imaged in spines before (basal) and after chem LTP in control and GluN1x-link conditions. Scale bar = 1 μm. Lower panel: CaMKII-GFP fluorescence intensity was compared between these conditions (basal: n = 6 neuronal fields, N = 765 spines; chem LTP: n = 11 neuronal fields, N = 1,688 spines; basal versus chem LTP, Student's t -test, *** P$
Figure Legend Snippet: The activity-dependent shift in CaMKII dynamics within dendritic spines is regulated by GluN2B-NMDAR dynamics Representative immunoblots showing the immunoprecipitation (IP) of CaMKII (α form) and phospho-CaMKII-Thr286 with GluN2B in membrane fractions from hippocampal slices (P17–20 rats) incubated with buffer (control) or GluN1 x-link. Lower panel: the ratio between CaMKII and GluN2B optical densities is represented ( n = 3 independent experiments). SM, start material; No Ab., no antibody; Cont., control. CaMKII-GFP was detected and imaged in spines before (basal) and after chem LTP in control and GluN1x-link conditions. Scale bar = 1 μm. Lower panel: CaMKII-GFP fluorescence intensity was compared between these conditions (basal: n = 6 neuronal fields, N = 765 spines; chem LTP: n = 11 neuronal fields, N = 1,688 spines; basal versus chem LTP, Student's t -test, *** P

Techniques Used: Activity Assay, Western Blot, Immunoprecipitation, Incubation, Fluorescence

Schematic model of the dynamic interplay between surface GluN2B-NMDAR and CaMKII during synaptic long-term potentiation (LTP) In basal conditions, GluN2B-NMDAR surface diffusion is regulated by the activity of CaMKII. During activity-dependent changes in glutamatergic synaptic transmission, increased surface dynamics of GluN2B-NMDAR favors the intracellular redistribution of CaMKII through their direct interaction, promoting the accumulation of CaMKII in spines.

Figure Legend Snippet: Schematic model of the dynamic interplay between surface GluN2B-NMDAR and CaMKII during synaptic long-term potentiation (LTP) In basal conditions, GluN2B-NMDAR surface diffusion is regulated by the activity of CaMKII. During activity-dependent changes in glutamatergic synaptic transmission, increased surface dynamics of GluN2B-NMDAR favors the intracellular redistribution of CaMKII through their direct interaction, promoting the accumulation of CaMKII in spines.

Techniques Used: Diffusion-based Assay, Activity Assay, Transmission Assay

The surface diffusion of synaptic GluN2B-NMDAR is rapidly increased after chem LTP in immature neurons Upper panels: Representative dendritic fragments of a hippocampal neuron (13 div) expressing GluA1-SEP and Homer 1c-DsRed. Clusters of GluA1-SEP were imaged for at least 30 min. The pseudocolor representation shows the different intensity levels of the GluA1-SEP staining. GluA1-SEP intensity was measured in synapses (Homer1c cluster) before and after the induction of chem LTP (see Materials and Methods). Note that 10 min after chem LTP, the GluA1-SEP fluorescence intensity increased in postsynaptic clusters. Arrows designate synapses (i.e., Homer). Bottom panels: magnifications and time-lapses of the outlined GluA1-SEP synaptic clusters. Scale bar = 5 μm; insets = 500 nm; bottom panels = 350 nm. Left panel: Representative example of a GluA1-SEP fluorescence time course before and after chem LTP (red) or buffer (black) protocol. The average GluA1-SEP synaptic intensity was significantly increased after chem LTP (24 min after induction) (middle panel). Student's t -test, *** P

Figure Legend Snippet: The surface diffusion of synaptic GluN2B-NMDAR is rapidly increased after chem LTP in immature neurons Upper panels: Representative dendritic fragments of a hippocampal neuron (13 div) expressing GluA1-SEP and Homer 1c-DsRed. Clusters of GluA1-SEP were imaged for at least 30 min. The pseudocolor representation shows the different intensity levels of the GluA1-SEP staining. GluA1-SEP intensity was measured in synapses (Homer1c cluster) before and after the induction of chem LTP (see Materials and Methods). Note that 10 min after chem LTP, the GluA1-SEP fluorescence intensity increased in postsynaptic clusters. Arrows designate synapses (i.e., Homer). Bottom panels: magnifications and time-lapses of the outlined GluA1-SEP synaptic clusters. Scale bar = 5 μm; insets = 500 nm; bottom panels = 350 nm. Left panel: Representative example of a GluA1-SEP fluorescence time course before and after chem LTP (red) or buffer (black) protocol. The average GluA1-SEP synaptic intensity was significantly increased after chem LTP (24 min after induction) (middle panel). Student's t -test, *** P

Techniques Used: Diffusion-based Assay, Expressing, Staining, Fluorescence

The surface cross-linking of NMDAR blocks chem LTP Comparison of the GluA1-SEP fluorescence within synapses (white dotted circle) in spines from control ( n = 786 synapses), GluN1 x-link ( n = 1324), or GluN2B x-link ( n = 987) condition. The dendritic shaft is indicated by the arrow head. Scale bar = 1 μm. The bar graphs represent the mean value ± s.e.m. Time-lapse imaging of spine areas containing GluA1-SEP (white dotted circle) from immature hippocampal neurons in control (no stimulation), chem LTP, chem LTP + GluN1 x-link, and chem LTP + GluN2B x-link conditions. The pseudocolor representation shows the different intensity levels of the GluA1-SEP staining. Scale bar = 1 μm. Comparison of the normalized GluA1-SEP fluorescence intensity within synapses in control ( n = 786 synapses), chem LTP alone ( n = 1324, *** P

Figure Legend Snippet: The surface cross-linking of NMDAR blocks chem LTP Comparison of the GluA1-SEP fluorescence within synapses (white dotted circle) in spines from control ( n = 786 synapses), GluN1 x-link ( n = 1324), or GluN2B x-link ( n = 987) condition. The dendritic shaft is indicated by the arrow head. Scale bar = 1 μm. The bar graphs represent the mean value ± s.e.m. Time-lapse imaging of spine areas containing GluA1-SEP (white dotted circle) from immature hippocampal neurons in control (no stimulation), chem LTP, chem LTP + GluN1 x-link, and chem LTP + GluN2B x-link conditions. The pseudocolor representation shows the different intensity levels of the GluA1-SEP staining. Scale bar = 1 μm. Comparison of the normalized GluA1-SEP fluorescence intensity within synapses in control ( n = 786 synapses), chem LTP alone ( n = 1324, *** P

Techniques Used: Fluorescence, Imaging, Staining

Surface cross-linking of NMDAR specifically impairs their surface diffusion without affecting their function Trajectories of single surface QD-GluN1-NMDAR (30-Hz acquisition, 30-s duration) in hippocampal neurons in control (left) and GluN1x-link (right) conditions. A schematic representation of the NMDAR x-link technique using primary anti-GluN (I ary Ab) and secondary (II ary Ab) antibodies is shown in the middle panel. Insets: enlarged GluN1-QD trajectories. Field scale bar = 5 μm; inset scale bar = 1 μm. Cumulative distribution of GluN1-NMDAR instantaneous surface diffusion coefficients in control and GluN1x-link conditions. Note the leftward shift of the curve in the GluN1x-link condition, indicating a slowdown of surface diffusion. Representative FRAP acquisition of GluA1-SEP in control and GluN1-NMDAR x-link conditions. The circles indicate the bleached regions. Scale bar = 5 μm. Average FRAP recovery curves of GluA1-SEP fluorescence in control ( n = 13), GluN1 x-link ( n = 3), and GluN2B x-link ( n = 4) conditions. Full lines represent the average recovery, while dotted lines represent the mean ± s.e.m. The fluorescence recovery of surface synaptic GluA1-SEP remained unaffected in all conditions ( P > 0.05). Representative images of a hippocampal neuron in the basal condition or after glutamate (30 μM) application. The pseudocolor representation shows the different intensity levels of the calcium indicator (Fluo4-AM, 2 μM) before and after the glutamate stimulation. Scale bar = 20 μm. Average calcium intensity change (ΔF/F0) over time after glutamate stimulation of hippocampal neurons ( n = 29). Relative comparison (percent of basal) of a transient calcium rise induced by glutamate in control ( n = 29), control + AP5 ( n = 12, Student's t -test, ** P

Figure Legend Snippet: Surface cross-linking of NMDAR specifically impairs their surface diffusion without affecting their function Trajectories of single surface QD-GluN1-NMDAR (30-Hz acquisition, 30-s duration) in hippocampal neurons in control (left) and GluN1x-link (right) conditions. A schematic representation of the NMDAR x-link technique using primary anti-GluN (I ary Ab) and secondary (II ary Ab) antibodies is shown in the middle panel. Insets: enlarged GluN1-QD trajectories. Field scale bar = 5 μm; inset scale bar = 1 μm. Cumulative distribution of GluN1-NMDAR instantaneous surface diffusion coefficients in control and GluN1x-link conditions. Note the leftward shift of the curve in the GluN1x-link condition, indicating a slowdown of surface diffusion. Representative FRAP acquisition of GluA1-SEP in control and GluN1-NMDAR x-link conditions. The circles indicate the bleached regions. Scale bar = 5 μm. Average FRAP recovery curves of GluA1-SEP fluorescence in control ( n = 13), GluN1 x-link ( n = 3), and GluN2B x-link ( n = 4) conditions. Full lines represent the average recovery, while dotted lines represent the mean ± s.e.m. The fluorescence recovery of surface synaptic GluA1-SEP remained unaffected in all conditions ( P > 0.05). Representative images of a hippocampal neuron in the basal condition or after glutamate (30 μM) application. The pseudocolor representation shows the different intensity levels of the calcium indicator (Fluo4-AM, 2 μM) before and after the glutamate stimulation. Scale bar = 20 μm. Average calcium intensity change (ΔF/F0) over time after glutamate stimulation of hippocampal neurons ( n = 29). Relative comparison (percent of basal) of a transient calcium rise induced by glutamate in control ( n = 29), control + AP5 ( n = 12, Student's t -test, ** P

Techniques Used: Diffusion-based Assay, Fluorescence

$Synaptic GluN2B-NMDAR are laterally redistributed in the synaptic area following chem LTP in immature neurons Detection of a single QD (30-Hz acquisition) in our experimental conditions. The high signal-to-noise ratio (SNR) ( > 5) enables the detection and location of the signal with a high pointing accuracy (˜30 nm). The QD fluorescence is quantified on a pseudocolor scale (low: red; high: yellow). Scale bar = 800 nm. A 500-frame stack is obtained while tracking down a single NMDAR/QD complex. On each frame, a single GluN2B- (green) or GluN2A- (blue) QD particle complex is detected and precisely located within synaptic (dark gray) and perisynaptic (320-nm annulus around the synapse; light gray) areas. Those 500 locations are then projected on a single image, providing the successive positions of this receptor/particle complex during the 500-frame stack. Note that GluN2A-NMDAR are more concentrated within the core of the PSD. Scale bar image = 300 nm; synaptic areas = 200 nm. Relative fraction of synaptic and perisynaptic GluN2-QD particles, calculated before and after chem LTP for both GluN2B- (left) and GluN2A-NMDAR (right) ( n = 25 and 20 dendritic fields before and after chem LTP, respectively). Note the significant decrease in the relative synaptic content in GluN2B-NMDAR particles right after chem LTP (Student's t -test, ** P$
Figure Legend Snippet: Synaptic GluN2B-NMDAR are laterally redistributed in the synaptic area following chem LTP in immature neurons Detection of a single QD (30-Hz acquisition) in our experimental conditions. The high signal-to-noise ratio (SNR) ( > 5) enables the detection and location of the signal with a high pointing accuracy (˜30 nm). The QD fluorescence is quantified on a pseudocolor scale (low: red; high: yellow). Scale bar = 800 nm. A 500-frame stack is obtained while tracking down a single NMDAR/QD complex. On each frame, a single GluN2B- (green) or GluN2A- (blue) QD particle complex is detected and precisely located within synaptic (dark gray) and perisynaptic (320-nm annulus around the synapse; light gray) areas. Those 500 locations are then projected on a single image, providing the successive positions of this receptor/particle complex during the 500-frame stack. Note that GluN2A-NMDAR are more concentrated within the core of the PSD. Scale bar image = 300 nm; synaptic areas = 200 nm. Relative fraction of synaptic and perisynaptic GluN2-QD particles, calculated before and after chem LTP for both GluN2B- (left) and GluN2A-NMDAR (right) ( n = 25 and 20 dendritic fields before and after chem LTP, respectively). Note the significant decrease in the relative synaptic content in GluN2B-NMDAR particles right after chem LTP (Student's t -test, ** P

Techniques Used: Fluorescence

4) Product Images from "Neuronal Plasticity in the Cingulate Cortex of Rats following Esophageal Acid Exposure in Early Life"

Article Title: Neuronal Plasticity in the Cingulate Cortex of Rats following Esophageal Acid Exposure in Early Life

Journal: Gastroenterology

doi: 10.1053/j.gastro.2011.04.044

NR2B expression pattern in rCCs at P60 following esophageal acid exposure at different time points of development. The treatment strategies were the same as indicated in . A. blots show NR2B and β-actin expression in individual animals

Figure Legend Snippet: NR2B expression pattern in rCCs at P60 following esophageal acid exposure at different time points of development. The treatment strategies were the same as indicated in . A. blots show NR2B and β-actin expression in individual animals

Techniques Used: Expressing

A : blots represent phosphorylation pattern of NR2B subunit in rCCs from naïve rats. B : blots show SerP1303 NR2B and β-actin expression in 4 groups of animals. The treatment strategies were the same as described in . C : bar graphs

Figure Legend Snippet: A : blots represent phosphorylation pattern of NR2B subunit in rCCs from naïve rats. B : blots show SerP1303 NR2B and β-actin expression in 4 groups of animals. The treatment strategies were the same as described in . C : bar graphs

Techniques Used: Expressing

5) Product Images from "Lack of the Actin Capping Protein, Eps8, Affects NMDA-Type Glutamate Receptor Function and Composition"

Article Title: Lack of the Actin Capping Protein, Eps8, Affects NMDA-Type Glutamate Receptor Function and Composition

Journal: Frontiers in Molecular Neuroscience

doi: 10.3389/fnmol.2018.00313

Techniques Used: Mass Spectrometry, Inhibition, MANN-WHITNEY, Concentration Assay, Quantitation Assay

Techniques Used: Western Blot

6) Product Images from "SAP97 Binding Partner CRIPT Promotes Dendrite Growth In Vitro and In Vivo"

Article Title: SAP97 Binding Partner CRIPT Promotes Dendrite Growth In Vitro and In Vivo

Journal: eNeuro

doi: 10.1523/ENEURO.0175-17.2017

CRIPT knockdown leads to a selective reduction in the abundance of GluA1 and SAP97. Mixed spinal cord cultures were infected with HSV engineered to express a miRNA targeting CRIPT or a scrambled control. Two days later, lysates were prepared and subjected to Western blottings. No more than six independent experiments were performed for the quantitative image analysis. CRIPT knockdown leads to a reduction in GluA1 and SAP97 abundance and no effect on the abundance of GluA2, GluA4, NR1, NR2A, NR2B, or PSD95. Representative images of Western blottings with actin loading controls are shown and quantification of band intensity in the bar graphs below; *significant difference between groups, p

Figure Legend Snippet: CRIPT knockdown leads to a selective reduction in the abundance of GluA1 and SAP97. Mixed spinal cord cultures were infected with HSV engineered to express a miRNA targeting CRIPT or a scrambled control. Two days later, lysates were prepared and subjected to Western blottings. No more than six independent experiments were performed for the quantitative image analysis. CRIPT knockdown leads to a reduction in GluA1 and SAP97 abundance and no effect on the abundance of GluA2, GluA4, NR1, NR2A, NR2B, or PSD95. Representative images of Western blottings with actin loading controls are shown and quantification of band intensity in the bar graphs below; *significant difference between groups, p

Techniques Used: Infection, Western Blot

7) Product Images from "Functional NMDA receptors are expressed by human pulmonary artery smooth muscle cells"

Article Title: Functional NMDA receptors are expressed by human pulmonary artery smooth muscle cells

Journal: Scientific Reports

doi: 10.1038/s41598-021-87667-0

Functional NMDA receptors are localized on the surface of HPASMCs. Immunofluorescence staining was performed on non-permeabilized HPASMCs. Both GluN1 and GluN2 (2B and 2D) subunits were detected on the surface of HPASMCs ( A , B ). GluN1 co-localized with GluN2B ( A ) and GluN2D ( B ) on the surface of HPASMCs, respectively. Results are representative images taken from at least three separate experiments. Scale bar = 100 µM. ( C , D ) are representative scatter plots of each fluorescent pixel from confocal images with GluN1 green fluorescent intensity along the x -axis and GluN2 red fluorescent intensity along the y -axis. The shaded area in the upper right quadrant represents colocalized pixels above background with the associated Pearson correlation coefficient indicated for all colocalized pixels. ( E ) Representative traces for cultured HPASMCs in response to 100 μM NMDA and 10 μM glycine treatment. Cells were clamped at − 50 mV and whole cell recordings were performed. ( F ) NMDA and glycine treatment evoked an inward whole-cell current of 10.9 ± 1.7 pA (Mean ± SE, **p

Figure Legend Snippet: Functional NMDA receptors are localized on the surface of HPASMCs. Immunofluorescence staining was performed on non-permeabilized HPASMCs. Both GluN1 and GluN2 (2B and 2D) subunits were detected on the surface of HPASMCs ( A , B ). GluN1 co-localized with GluN2B ( A ) and GluN2D ( B ) on the surface of HPASMCs, respectively. Results are representative images taken from at least three separate experiments. Scale bar = 100 µM. ( C , D ) are representative scatter plots of each fluorescent pixel from confocal images with GluN1 green fluorescent intensity along the x -axis and GluN2 red fluorescent intensity along the y -axis. The shaded area in the upper right quadrant represents colocalized pixels above background with the associated Pearson correlation coefficient indicated for all colocalized pixels. ( E ) Representative traces for cultured HPASMCs in response to 100 μM NMDA and 10 μM glycine treatment. Cells were clamped at − 50 mV and whole cell recordings were performed. ( F ) NMDA and glycine treatment evoked an inward whole-cell current of 10.9 ± 1.7 pA (Mean ± SE, **p

Techniques Used: Functional Assay, Immunofluorescence, Staining, Cell Culture

8) Product Images from "CXCL12 inhibits expression of the NMDA receptor's NR2B subunit through a histone deacetylase-dependent pathway contributing to neuronal survival"

Article Title: CXCL12 inhibits expression of the NMDA receptor's NR2B subunit through a histone deacetylase-dependent pathway contributing to neuronal survival

Journal: Cell Death & Disease

doi: 10.1038/cddis.2010.10

In vivo AMD3100 administration increases NR2B protein levels in the rat cortex. ( a ) AMD3100 treatment decreases CXCR4 phosphorylation in brain slices of treated animals as detected through immunohistochemistry, using phospho-specific antibodies against ligand-activated CXCR4. Three animals per group were analyzed and no changes were observed in total levels of CXCR4. ( b ) Studies in homogenized tissue samples (cerebral cortex and hippocampus) also show a reduction in phosphorylated levels of CXCR4 compared with total CXCR4 ( * P

Figure Legend Snippet: In vivo AMD3100 administration increases NR2B protein levels in the rat cortex. ( a ) AMD3100 treatment decreases CXCR4 phosphorylation in brain slices of treated animals as detected through immunohistochemistry, using phospho-specific antibodies against ligand-activated CXCR4. Three animals per group were analyzed and no changes were observed in total levels of CXCR4. ( b ) Studies in homogenized tissue samples (cerebral cortex and hippocampus) also show a reduction in phosphorylated levels of CXCR4 compared with total CXCR4 ( * P

Techniques Used: In Vivo, Immunohistochemistry

CXCL12 treatment reduces levels of NR2B protein and mRNA but does not alter other NMDA subunits. ( a ) Addition of CXCL12 (20 nM, 1–24 h) to neuronal culture media decreases NR2B protein levels in a time-dependent manner. Graph shows data from three independent experiments ( * P

Figure Legend Snippet: CXCL12 treatment reduces levels of NR2B protein and mRNA but does not alter other NMDA subunits. ( a ) Addition of CXCL12 (20 nM, 1–24 h) to neuronal culture media decreases NR2B protein levels in a time-dependent manner. Graph shows data from three independent experiments ( * P

Techniques Used:

CXCL12 reduces global histone H3 acetylation in neurons, and histone deacetylase (HDAC) inhibitors prevent the effects of CXCL12 on the NR2B. ( a ) Global H3 acetylation levels were measured through a colorimetric acetylation assay as indicated in the ‘Materials and methods' section. Reduced levels of histone acetylation were found in CXCL12-treated (20 nM) neurons compared with control; this effect is blocked by cotreatment with TSA (100 nM) ( * P

Figure Legend Snippet: CXCL12 reduces global histone H3 acetylation in neurons, and histone deacetylase (HDAC) inhibitors prevent the effects of CXCL12 on the NR2B. ( a ) Global H3 acetylation levels were measured through a colorimetric acetylation assay as indicated in the ‘Materials and methods' section. Reduced levels of histone acetylation were found in CXCL12-treated (20 nM) neurons compared with control; this effect is blocked by cotreatment with TSA (100 nM) ( * P

Techniques Used: Histone Deacetylase Assay, Acetylation Assay

9) Product Images from "Fingolimod Limits Acute Aβ Neurotoxicity and Promotes Synaptic Versus Extrasynaptic NMDA Receptor Functionality in Hippocampal Neurons"

Article Title: Fingolimod Limits Acute Aβ Neurotoxicity and Promotes Synaptic Versus Extrasynaptic NMDA Receptor Functionality in Hippocampal Neurons

Journal: Scientific Reports

doi: 10.1038/srep41734

$FTY720 modulates GLUN2B localization in the postsynaptic fraction and increases surface GLUN2B and GLUN2A expression at synapses. ( A , B ) Western blotting of the homogenate (HOMO), postsynaptic triton insoluble fraction (TIF) and triton soluble fraction (TSF) obtained from control and FTY720-treated hippocampal neurons (DIV14). Tubulin is used as loading control, while PSD-95 as a marker of postsynaptic fraction. FTY720 leads to an increased GLUN2B localization in the TIF leaving the total amount of GLUN2B unaltered while doesn’t alter GLUN2A localization in TIF (Student’s t-test, P = 0.0275 N = 4). ( C ) Confocal images of neurons live stained for GLUN2A, fixed and counterstained against β3-tubulin. Relative quantification of density of GLUN2A puncta are shown on the left (Mann-Whitney Rank Sum Test, P = 0.070) and mean intensity of GLUN2A clusters on the right (Mann-Whitney Rank Sum Test, P = 0.001). ( D ) Images of neurons live stained for GLUN2B and quantification of GLUN2B puncta density (Mann-Whitney Rank Sum Test, P = 0.003) and mean intensity (Student’s t-test, P = 0.610). ( E , F ) Images of neurons live stained for GLUN2A ( E ) or GLUN2B ( F ), fixed and counterstained against the presynaptic marker bassoon. Right histograms show quantification of GLUN2A positive synapses (Mann-Whitney Rank Sum Test, P ≤ 0.001) and GLUN2A puncta mean intensity (Mann-Whitney Rank Sum Test, P = 0.005) (number of analyzed puncta: control = 1010, FTY720 = 720; number of analyzed fields: control = 35; FTY720 = 30) or GLUN2B positive synapses (Mann-Whitney Rank Sum Test, P = 0.005) and GLUN2B puncta mean intensity (number of analyzed puncta: control = 1250, FTY720 = 807; number of analyzed fields: control = 37; FTY720 = 32). ( G ) Representative images of spines co-transfected with SEP-GluN2A(green) and dTom (red) acquired at the indicated time point in control and FTY720-treated neurons. ( H ) XY graph representing the DMFI/MFI 0 of SEP-GluN2A over time (number of analysed spines: control = 40, FTY720 = 47; Mann-Whitney Rank Sum Test, P = 0.037 at t 50 and P = 0.009 at t 60 ). ( I ) Images of spines co-transfected with SEP-GluN2B and dTom as in G. ( J ) XY graph representing the DMFI/MFI 0 of SEP-GluN2B over time (number of analysed spines: control = 15, FTY720 = 22; Mann-Whitney Rank Sum Test, P = 0.030 at t 60 ).$
Figure Legend Snippet: FTY720 modulates GLUN2B localization in the postsynaptic fraction and increases surface GLUN2B and GLUN2A expression at synapses. ( A , B ) Western blotting of the homogenate (HOMO), postsynaptic triton insoluble fraction (TIF) and triton soluble fraction (TSF) obtained from control and FTY720-treated hippocampal neurons (DIV14). Tubulin is used as loading control, while PSD-95 as a marker of postsynaptic fraction. FTY720 leads to an increased GLUN2B localization in the TIF leaving the total amount of GLUN2B unaltered while doesn’t alter GLUN2A localization in TIF (Student’s t-test, P = 0.0275 N = 4). ( C ) Confocal images of neurons live stained for GLUN2A, fixed and counterstained against β3-tubulin. Relative quantification of density of GLUN2A puncta are shown on the left (Mann-Whitney Rank Sum Test, P = 0.070) and mean intensity of GLUN2A clusters on the right (Mann-Whitney Rank Sum Test, P = 0.001). ( D ) Images of neurons live stained for GLUN2B and quantification of GLUN2B puncta density (Mann-Whitney Rank Sum Test, P = 0.003) and mean intensity (Student’s t-test, P = 0.610). ( E , F ) Images of neurons live stained for GLUN2A ( E ) or GLUN2B ( F ), fixed and counterstained against the presynaptic marker bassoon. Right histograms show quantification of GLUN2A positive synapses (Mann-Whitney Rank Sum Test, P ≤ 0.001) and GLUN2A puncta mean intensity (Mann-Whitney Rank Sum Test, P = 0.005) (number of analyzed puncta: control = 1010, FTY720 = 720; number of analyzed fields: control = 35; FTY720 = 30) or GLUN2B positive synapses (Mann-Whitney Rank Sum Test, P = 0.005) and GLUN2B puncta mean intensity (number of analyzed puncta: control = 1250, FTY720 = 807; number of analyzed fields: control = 37; FTY720 = 32). ( G ) Representative images of spines co-transfected with SEP-GluN2A(green) and dTom (red) acquired at the indicated time point in control and FTY720-treated neurons. ( H ) XY graph representing the DMFI/MFI 0 of SEP-GluN2A over time (number of analysed spines: control = 40, FTY720 = 47; Mann-Whitney Rank Sum Test, P = 0.037 at t 50 and P = 0.009 at t 60 ). ( I ) Images of spines co-transfected with SEP-GluN2B and dTom as in G. ( J ) XY graph representing the DMFI/MFI 0 of SEP-GluN2B over time (number of analysed spines: control = 15, FTY720 = 22; Mann-Whitney Rank Sum Test, P = 0.030 at t 60 ).

Techniques Used: Expressing, Western Blot, Marker, Staining, MANN-WHITNEY, Transfection

glun2b (Alomone Labs)

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2) Product Images from "Lack of the Actin Capping Protein, Eps8, Affects NMDA-Type Glutamate Receptor Function and Composition"

3) Product Images from "Surface dynamics of GluN2B-NMDA receptors controls plasticity of maturing glutamate synapses"

4) Product Images from "Neuronal Plasticity in the Cingulate Cortex of Rats following Esophageal Acid Exposure in Early Life"

5) Product Images from "Lack of the Actin Capping Protein, Eps8, Affects NMDA-Type Glutamate Receptor Function and Composition"

6) Product Images from "SAP97 Binding Partner CRIPT Promotes Dendrite Growth In Vitro and In Vivo"

7) Product Images from "Functional NMDA receptors are expressed by human pulmonary artery smooth muscle cells"

8) Product Images from "CXCL12 inhibits expression of the NMDA receptor's NR2B subunit through a histone deacetylase-dependent pathway contributing to neuronal survival"

9) Product Images from "Fingolimod Limits Acute Aβ Neurotoxicity and Promotes Synaptic Versus Extrasynaptic NMDA Receptor Functionality in Hippocampal Neurons"

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