glun1  (Alomone Labs)


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    Structured Review

    Alomone Labs glun1
    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 <t>anti-GluN1</t> 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
    Glun1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) 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

    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

    Acute GluN1-NMDAR x-link prevents LTP at CA3-CA1 synapse in rat hippocampal slices A–C Slices from P15 to 20 Wistar rats were incubated in a regular (control) or anti-GluN1-supplemented ACSF (x-link GluN1) for 45 min prior to fEPSP recordings (A). Average field responses in various conditions are represented (B). A robust LTP, as measured by the slope of the fEPSP, was induced by five trains of 20 pulses at 100 Hz (C). LTP amplitude decreased over the first 5 min before reaching a stable plateau. Cross-linking the GluN1 subunits with antibody substantially decreased LTP amplitude. There was no difference in fEPSP slope between control ( n = 8) and x-link GluN1 ( n = 8) under basal condition ( P > 0.05). Recordings without LTP-inducing trains either in GluN1 x-link or in control ASCF did not change over time. D Mean normalized fEPSP slope after 100-Hz stimulation in control ( n = 8; open bar) and GluN1 x-link ( n = 8; red bar) conditions from P15 to 20 (Student's t -test, * P
    Figure Legend Snippet: Acute GluN1-NMDAR x-link prevents LTP at CA3-CA1 synapse in rat hippocampal slices A–C Slices from P15 to 20 Wistar rats were incubated in a regular (control) or anti-GluN1-supplemented ACSF (x-link GluN1) for 45 min prior to fEPSP recordings (A). Average field responses in various conditions are represented (B). A robust LTP, as measured by the slope of the fEPSP, was induced by five trains of 20 pulses at 100 Hz (C). LTP amplitude decreased over the first 5 min before reaching a stable plateau. Cross-linking the GluN1 subunits with antibody substantially decreased LTP amplitude. There was no difference in fEPSP slope between control ( n = 8) and x-link GluN1 ( n = 8) under basal condition ( P > 0.05). Recordings without LTP-inducing trains either in GluN1 x-link or in control ASCF did not change over time. D Mean normalized fEPSP slope after 100-Hz stimulation in control ( n = 8; open bar) and GluN1 x-link ( n = 8; red bar) conditions from P15 to 20 (Student's t -test, * P

    Techniques Used: Incubation

    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

    2) Product Images from "Stress hormone rapidly tunes synaptic NMDA receptor through membrane dynamics and mineralocorticoid signalling"

    Article Title: Stress hormone rapidly tunes synaptic NMDA receptor through membrane dynamics and mineralocorticoid signalling

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-08695-3

    Corticosterone-induced AMPAR synaptic increase is prevented by surface NMDAR cross-linking (x-link). ( A ) Schematic representation of the experimental design ( a 1 ). Characteristic effect of GluN1 cross-linking on GluN1-NMDAR surface diffusion. Note the strong reduction in trajectory lengths in GluN1 x-link (20 min exposure) when compared to the control IgG condition ( a 2 ). Scale bar, 4 µm. ( B ) Representative distributions of GluA1-AMPAR within synapses exposed to corticosterone alone or corticosterone plus GluN1 x-link. Scale bar, 500 nm. ( C ) Comparison of the percent of synaptic GluA1-AMPAR over the extrasynaptic ones between conditions (control, n = 26 dendritic fields; corticoterone, n = 28 dendritic fields; corticosterone + GluN1 x-link, n = 32 dendritic fields; N > 7 neurons for each condition). *p
    Figure Legend Snippet: Corticosterone-induced AMPAR synaptic increase is prevented by surface NMDAR cross-linking (x-link). ( A ) Schematic representation of the experimental design ( a 1 ). Characteristic effect of GluN1 cross-linking on GluN1-NMDAR surface diffusion. Note the strong reduction in trajectory lengths in GluN1 x-link (20 min exposure) when compared to the control IgG condition ( a 2 ). Scale bar, 4 µm. ( B ) Representative distributions of GluA1-AMPAR within synapses exposed to corticosterone alone or corticosterone plus GluN1 x-link. Scale bar, 500 nm. ( C ) Comparison of the percent of synaptic GluA1-AMPAR over the extrasynaptic ones between conditions (control, n = 26 dendritic fields; corticoterone, n = 28 dendritic fields; corticosterone + GluN1 x-link, n = 32 dendritic fields; N > 7 neurons for each condition). *p

    Techniques Used: Diffusion-based Assay

    Corticosterone decreases the surface dynamics of GluN1-NMDAR in hippocampal neurons exposed to corticosterone. ( A ) Schematic representation of antibody against GluN1 subunit and single QD complex used to label and track surface NMDAR. ( B ) Representative trajectories of single GluN1-NMDAR in control (buffer, blue) and corticosterone (100 nM, 20 min; red). Note that the traces represent different receptors. The black arrows point toward spines in which glutamatergic synapses were identified. Lower panels, enlarged trajectories located within the postsynaptic densities (gray areas). Starting and ending time of the single trajectories are indicated as for instance time 0 (t 0s ). ( C ) Comparison of the synaptic dwell-time (expressed in seconds) of surface GluN1-NMDAR in buffer (n = 55 trajectories) or corticosterone (n = 62 trajectories) condition. ***p
    Figure Legend Snippet: Corticosterone decreases the surface dynamics of GluN1-NMDAR in hippocampal neurons exposed to corticosterone. ( A ) Schematic representation of antibody against GluN1 subunit and single QD complex used to label and track surface NMDAR. ( B ) Representative trajectories of single GluN1-NMDAR in control (buffer, blue) and corticosterone (100 nM, 20 min; red). Note that the traces represent different receptors. The black arrows point toward spines in which glutamatergic synapses were identified. Lower panels, enlarged trajectories located within the postsynaptic densities (gray areas). Starting and ending time of the single trajectories are indicated as for instance time 0 (t 0s ). ( C ) Comparison of the synaptic dwell-time (expressed in seconds) of surface GluN1-NMDAR in buffer (n = 55 trajectories) or corticosterone (n = 62 trajectories) condition. ***p

    Techniques Used:

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    Alomone Labs rabbit anti glun1
    Distribution of <t>GluN1</t> immunoreactivity in young and aged GIN mice hippocampus. (A1,B1) Panoramic confocal planes showing the distribution of O-LM cells (green) and GluN1 immunoreactivity (red) in the hippocampus of 3-month-old (A1) and 16-month-old (B1) mice. Different regions and strata are indicated with dotted lines. (A2,B2) High magnification view from the different CA1 strata in 3-month-old (A2) and 16-month-old (B2) mice. (A3,B3) Enlarged view of the squared regions in panels (A2,B2) , showing double immunofluorescence for GFP/GluN1, in strata oriens , and pyramidale. Note the homogenous distribution of GluN1 immunoreactive puncta in pyramidal neurons in both 3-month-old (A3) and 16-month-old (B3) mice. Scale bar: 150 μm for panels (A1,B1) , 67 μm for panels (A2,B2) , and 21 μm for panels (A3,B3) .
    Rabbit Anti Glun1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti glun1/product/Alomone Labs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    94
    Alomone Labs glun1
    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 <t>anti-GluN1</t> 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
    Glun1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/glun1/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    glun1 - by Bioz Stars, 2022-08
    94/100 stars
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    Image Search Results


    Distribution of GluN1 immunoreactivity in young and aged GIN mice hippocampus. (A1,B1) Panoramic confocal planes showing the distribution of O-LM cells (green) and GluN1 immunoreactivity (red) in the hippocampus of 3-month-old (A1) and 16-month-old (B1) mice. Different regions and strata are indicated with dotted lines. (A2,B2) High magnification view from the different CA1 strata in 3-month-old (A2) and 16-month-old (B2) mice. (A3,B3) Enlarged view of the squared regions in panels (A2,B2) , showing double immunofluorescence for GFP/GluN1, in strata oriens , and pyramidale. Note the homogenous distribution of GluN1 immunoreactive puncta in pyramidal neurons in both 3-month-old (A3) and 16-month-old (B3) mice. Scale bar: 150 μm for panels (A1,B1) , 67 μm for panels (A2,B2) , and 21 μm for panels (A3,B3) .

    Journal: Frontiers in Aging Neuroscience

    Article Title: Effects of Aging on the Structure and Expression of NMDA Receptors of Somatostatin Expressing Neurons in the Mouse Hippocampus

    doi: 10.3389/fnagi.2021.782737

    Figure Lengend Snippet: Distribution of GluN1 immunoreactivity in young and aged GIN mice hippocampus. (A1,B1) Panoramic confocal planes showing the distribution of O-LM cells (green) and GluN1 immunoreactivity (red) in the hippocampus of 3-month-old (A1) and 16-month-old (B1) mice. Different regions and strata are indicated with dotted lines. (A2,B2) High magnification view from the different CA1 strata in 3-month-old (A2) and 16-month-old (B2) mice. (A3,B3) Enlarged view of the squared regions in panels (A2,B2) , showing double immunofluorescence for GFP/GluN1, in strata oriens , and pyramidale. Note the homogenous distribution of GluN1 immunoreactive puncta in pyramidal neurons in both 3-month-old (A3) and 16-month-old (B3) mice. Scale bar: 150 μm for panels (A1,B1) , 67 μm for panels (A2,B2) , and 21 μm for panels (A3,B3) .

    Article Snippet: In order to study the GluN1 expression, sections were incubated with rabbit anti-GluN1 (Alomone, 1:400) or GluN2B rabbit anti-GluN2B (Alomone, 1:4000) together with chicken anti-GFP IgY (Abcam, 1:500) primary antibodies for 48 h at 4°C.

    Techniques: Mouse Assay, Immunofluorescence

    Analysis of the density and percentage of area covered with GluN1 immunoreactive puncta in the somata and the periphery of O-LM cells during aging. (A–F) Double GFP/GluN1 immunohistochemistry in 3-month-old (A) , 9-month-old (B) and 16-month-old (C) female mice, and in 3-month-old (D) , 9-month-old (E) and 16-month-old (F) male mice. (G–I) Graphs showing the density and percentage of area covered with GluN1 immunoreactive puncta in the somata (G1,G2–I2) and in its periphery (G3,G4–I4) in animals segregated by sex (G1–4) , pooled females (H1–4) and males (I1–4) (all graphs represent mean ± SEM). Scale bar: 5 μm.

    Journal: Frontiers in Aging Neuroscience

    Article Title: Effects of Aging on the Structure and Expression of NMDA Receptors of Somatostatin Expressing Neurons in the Mouse Hippocampus

    doi: 10.3389/fnagi.2021.782737

    Figure Lengend Snippet: Analysis of the density and percentage of area covered with GluN1 immunoreactive puncta in the somata and the periphery of O-LM cells during aging. (A–F) Double GFP/GluN1 immunohistochemistry in 3-month-old (A) , 9-month-old (B) and 16-month-old (C) female mice, and in 3-month-old (D) , 9-month-old (E) and 16-month-old (F) male mice. (G–I) Graphs showing the density and percentage of area covered with GluN1 immunoreactive puncta in the somata (G1,G2–I2) and in its periphery (G3,G4–I4) in animals segregated by sex (G1–4) , pooled females (H1–4) and males (I1–4) (all graphs represent mean ± SEM). Scale bar: 5 μm.

    Article Snippet: In order to study the GluN1 expression, sections were incubated with rabbit anti-GluN1 (Alomone, 1:400) or GluN2B rabbit anti-GluN2B (Alomone, 1:4000) together with chicken anti-GFP IgY (Abcam, 1:500) primary antibodies for 48 h at 4°C.

    Techniques: Immunohistochemistry, Mouse Assay

    Exposure to high glucose (HG) increases expression of NMDA receptor subunits in primary cultures of rat mesangial cells. A : Representative results of RT-PCR showing significantly increased abundance of transcripts encoding NR1, NR2B, and NR2C subunits but not of NR2A or NR2D in cells cultured for 24 h in HG medium compared with cells cultured in normal glucose (control [Con]). B : Densitometric analysis of three repetitions of the experiments shown in A . C : Immunoblot analysis showing increased abundance of NMDA receptor subunits in primary cultures of rat mesangial cells cultured in HG. D : Densitometric analysis of three repetitions of the experiments shown in C . Data are mean ± SD. * P

    Journal: Diabetes

    Article Title: NMDA Receptors as Potential Therapeutic Targets in Diabetic Nephropathy: Increased Renal NMDA Receptor Subunit Expression in Akita Mice and Reduced Nephropathy Following Sustained Treatment With Memantine or MK-801

    doi: 10.2337/db16-0209

    Figure Lengend Snippet: Exposure to high glucose (HG) increases expression of NMDA receptor subunits in primary cultures of rat mesangial cells. A : Representative results of RT-PCR showing significantly increased abundance of transcripts encoding NR1, NR2B, and NR2C subunits but not of NR2A or NR2D in cells cultured for 24 h in HG medium compared with cells cultured in normal glucose (control [Con]). B : Densitometric analysis of three repetitions of the experiments shown in A . C : Immunoblot analysis showing increased abundance of NMDA receptor subunits in primary cultures of rat mesangial cells cultured in HG. D : Densitometric analysis of three repetitions of the experiments shown in C . Data are mean ± SD. * P

    Article Snippet: Primary antibodies were rabbit anti-NR1 (AGC-001 1:100; Alomone Labs) or rabbit anti-NR2A (AGC-002 1:100; Alomone Labs) for 24 h at 4°C.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture

    Increased expression of NMDA receptor subunits in renal cortex of Akita mice. A : Representative results of RT-PCR showing increased abundance of transcripts encoding NR1, NR2A, and NR2C subunits but not in NR2B or NR2D in renal cortex in 12-week-old Akita mice compared with 12-week-old DBA/2J control mice. B : Densitometric analysis from four mice per group. C : Immunoblot analysis showing increased abundance of NMDA receptor subunits in Akita mice compared with DBA/2J control mice. D : Densitometric analysis from four mice per group. Data are mean ± SD. * P

    Journal: Diabetes

    Article Title: NMDA Receptors as Potential Therapeutic Targets in Diabetic Nephropathy: Increased Renal NMDA Receptor Subunit Expression in Akita Mice and Reduced Nephropathy Following Sustained Treatment With Memantine or MK-801

    doi: 10.2337/db16-0209

    Figure Lengend Snippet: Increased expression of NMDA receptor subunits in renal cortex of Akita mice. A : Representative results of RT-PCR showing increased abundance of transcripts encoding NR1, NR2A, and NR2C subunits but not in NR2B or NR2D in renal cortex in 12-week-old Akita mice compared with 12-week-old DBA/2J control mice. B : Densitometric analysis from four mice per group. C : Immunoblot analysis showing increased abundance of NMDA receptor subunits in Akita mice compared with DBA/2J control mice. D : Densitometric analysis from four mice per group. Data are mean ± SD. * P

    Article Snippet: Primary antibodies were rabbit anti-NR1 (AGC-001 1:100; Alomone Labs) or rabbit anti-NR2A (AGC-002 1:100; Alomone Labs) for 24 h at 4°C.

    Techniques: Expressing, Mouse Assay, Reverse Transcription Polymerase Chain Reaction

    Immunohistochemistry (IHC) suggests increased abundance of NMDA receptor subunits throughout the kidney of 12-week-old Akita mice. IHC was carried out in paraffin sections. Negative control sections shown at the top were not exposed to a primary antibody. A : Especially large increases in NR1, NR2A, and NR2C in renal tubules. B : Signal in glomeruli for NR1, NR2A, and NR2C. Primary processes were visible in some of the cells within glomeruli. C : Staining intensity per square micron in the whole kidney (top) and within glomeruli (bottom). Data are mean ± SD. * P

    Journal: Diabetes

    Article Title: NMDA Receptors as Potential Therapeutic Targets in Diabetic Nephropathy: Increased Renal NMDA Receptor Subunit Expression in Akita Mice and Reduced Nephropathy Following Sustained Treatment With Memantine or MK-801

    doi: 10.2337/db16-0209

    Figure Lengend Snippet: Immunohistochemistry (IHC) suggests increased abundance of NMDA receptor subunits throughout the kidney of 12-week-old Akita mice. IHC was carried out in paraffin sections. Negative control sections shown at the top were not exposed to a primary antibody. A : Especially large increases in NR1, NR2A, and NR2C in renal tubules. B : Signal in glomeruli for NR1, NR2A, and NR2C. Primary processes were visible in some of the cells within glomeruli. C : Staining intensity per square micron in the whole kidney (top) and within glomeruli (bottom). Data are mean ± SD. * P

    Article Snippet: Primary antibodies were rabbit anti-NR1 (AGC-001 1:100; Alomone Labs) or rabbit anti-NR2A (AGC-002 1:100; Alomone Labs) for 24 h at 4°C.

    Techniques: Immunohistochemistry, Mouse Assay, Negative Control, Staining

    Exposure to high glucose (HG) increases expression of NMDA receptor subunits in cultured mouse podocytes (MPC-5 cells). A : Representative results of RT-PCR showing increased abundance of transcripts encoding NR1, NR2A, NR2B, and NR2C subunits in cells cultured for 24 h in a medium containing 25 mmol/L glucose (HG). There was no change in NR2D. Control cells (Con) were cultured in medium containing 9 mmol/L glucose, with 16 mmol/L mannitol as an osmotic control. B : Densitometric analysis of three repetitions of the experiments shown in A . C : Immunoblot analysis showing increased abundance of NMDA receptor subunits in podocytes cultured in HG compared with Con. D : Densitometric analysis of three repetitions of the experiments shown in C . Data are mean ± SD. * P

    Journal: Diabetes

    Article Title: NMDA Receptors as Potential Therapeutic Targets in Diabetic Nephropathy: Increased Renal NMDA Receptor Subunit Expression in Akita Mice and Reduced Nephropathy Following Sustained Treatment With Memantine or MK-801

    doi: 10.2337/db16-0209

    Figure Lengend Snippet: Exposure to high glucose (HG) increases expression of NMDA receptor subunits in cultured mouse podocytes (MPC-5 cells). A : Representative results of RT-PCR showing increased abundance of transcripts encoding NR1, NR2A, NR2B, and NR2C subunits in cells cultured for 24 h in a medium containing 25 mmol/L glucose (HG). There was no change in NR2D. Control cells (Con) were cultured in medium containing 9 mmol/L glucose, with 16 mmol/L mannitol as an osmotic control. B : Densitometric analysis of three repetitions of the experiments shown in A . C : Immunoblot analysis showing increased abundance of NMDA receptor subunits in podocytes cultured in HG compared with Con. D : Densitometric analysis of three repetitions of the experiments shown in C . Data are mean ± SD. * P

    Article Snippet: Primary antibodies were rabbit anti-NR1 (AGC-001 1:100; Alomone Labs) or rabbit anti-NR2A (AGC-002 1:100; Alomone Labs) for 24 h at 4°C.

    Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction

    The expression of NMDAR in cortex was reduced in Emx1 cre/+ ; NR1 fl/fl mice. Examples of 12-μm coronal brain sections from P8 Emx1 cre/+ ; NR1 wt/wt (A) and Emx1 cre/+ ; NR1 fl/fl (B) of the same litter. Immunostaining of vesicular glutamate transporter 2 (VGult2) showed thalamocortical barrels in Layer IV of S1 which are pointed out by arrows. The VGlut2 staining in Emx1 cre/+ ; NR1 wt/wt mice revealed a clear barrel pattern (Aa). However, the barrel pattern in Emx1 cre/+ ; NR1 fl/fl mice was disrupted and less distinct (Ba). The NR1 staining in Emx1 cre/+ ; NR1 wt/wt mice were dense and strong in cortex (Ab, Ac). However, the staining in Emx1 cre/+ ; NR1 fl/fl mice was less bright and apparently reduced in Layer V and VI (Bb, Bc). Scale bar: 100μm for Ac and Bc; 500μm for rest of images.

    Journal: bioRxiv

    Article Title: NMDA receptors control cortical axonal projections via EPHRIN-B/EPHB signaling

    doi: 10.1101/2020.06.03.130559

    Figure Lengend Snippet: The expression of NMDAR in cortex was reduced in Emx1 cre/+ ; NR1 fl/fl mice. Examples of 12-μm coronal brain sections from P8 Emx1 cre/+ ; NR1 wt/wt (A) and Emx1 cre/+ ; NR1 fl/fl (B) of the same litter. Immunostaining of vesicular glutamate transporter 2 (VGult2) showed thalamocortical barrels in Layer IV of S1 which are pointed out by arrows. The VGlut2 staining in Emx1 cre/+ ; NR1 wt/wt mice revealed a clear barrel pattern (Aa). However, the barrel pattern in Emx1 cre/+ ; NR1 fl/fl mice was disrupted and less distinct (Ba). The NR1 staining in Emx1 cre/+ ; NR1 wt/wt mice were dense and strong in cortex (Ab, Ac). However, the staining in Emx1 cre/+ ; NR1 fl/fl mice was less bright and apparently reduced in Layer V and VI (Bb, Bc). Scale bar: 100μm for Ac and Bc; 500μm for rest of images.

    Article Snippet: Antibodies for immunostaining : Rabbit anti-NR1 (1:500, AGC-001, Alomone labs), anti-vGlut2 (1:200, AB2251, Millipore), goat anti-EphB2 (1:50, AF467, R & D), anti-cleaved caspase-3 ( # 9661S, Cell Signaling), anti-Rabbit 594 (#711-585-152, Jackson ImmunoResearch), and anti-guinea pig 488 (A-11073, Invitrogen).

    Techniques: Expressing, Mouse Assay, Immunostaining, Staining

    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

    Journal: The EMBO Journal

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

    doi: 10.1002/embj.201386356

    Figure Lengend 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

    Article Snippet: As previously described (Groc et al , ; Heine et al , ), for the x-link experiments, neurons were co-transfected with GluN1-SEP or GluN2B-SEP and Homer 1c-DsRed and incubated with highly concentrated (1:20) polyclonal antibodies directed against GluN1 (Alomone Labs; epitope corresponding to residues 385–399 of the GluN1 subunit), GluN2B (Alomone Labs; same as above), GluN2A (Alomone Labs; same as above) NMDAR subunits or against GFP (Chemicon).

    Techniques: 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

    Journal: The EMBO Journal

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

    doi: 10.1002/embj.201386356

    Figure Lengend 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

    Article Snippet: As previously described (Groc et al , ; Heine et al , ), for the x-link experiments, neurons were co-transfected with GluN1-SEP or GluN2B-SEP and Homer 1c-DsRed and incubated with highly concentrated (1:20) polyclonal antibodies directed against GluN1 (Alomone Labs; epitope corresponding to residues 385–399 of the GluN1 subunit), GluN2B (Alomone Labs; same as above), GluN2A (Alomone Labs; same as above) NMDAR subunits or against GFP (Chemicon).

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

    Acute GluN1-NMDAR x-link prevents LTP at CA3-CA1 synapse in rat hippocampal slices A–C Slices from P15 to 20 Wistar rats were incubated in a regular (control) or anti-GluN1-supplemented ACSF (x-link GluN1) for 45 min prior to fEPSP recordings (A). Average field responses in various conditions are represented (B). A robust LTP, as measured by the slope of the fEPSP, was induced by five trains of 20 pulses at 100 Hz (C). LTP amplitude decreased over the first 5 min before reaching a stable plateau. Cross-linking the GluN1 subunits with antibody substantially decreased LTP amplitude. There was no difference in fEPSP slope between control ( n = 8) and x-link GluN1 ( n = 8) under basal condition ( P > 0.05). Recordings without LTP-inducing trains either in GluN1 x-link or in control ASCF did not change over time. D Mean normalized fEPSP slope after 100-Hz stimulation in control ( n = 8; open bar) and GluN1 x-link ( n = 8; red bar) conditions from P15 to 20 (Student's t -test, * P

    Journal: The EMBO Journal

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

    doi: 10.1002/embj.201386356

    Figure Lengend Snippet: Acute GluN1-NMDAR x-link prevents LTP at CA3-CA1 synapse in rat hippocampal slices A–C Slices from P15 to 20 Wistar rats were incubated in a regular (control) or anti-GluN1-supplemented ACSF (x-link GluN1) for 45 min prior to fEPSP recordings (A). Average field responses in various conditions are represented (B). A robust LTP, as measured by the slope of the fEPSP, was induced by five trains of 20 pulses at 100 Hz (C). LTP amplitude decreased over the first 5 min before reaching a stable plateau. Cross-linking the GluN1 subunits with antibody substantially decreased LTP amplitude. There was no difference in fEPSP slope between control ( n = 8) and x-link GluN1 ( n = 8) under basal condition ( P > 0.05). Recordings without LTP-inducing trains either in GluN1 x-link or in control ASCF did not change over time. D Mean normalized fEPSP slope after 100-Hz stimulation in control ( n = 8; open bar) and GluN1 x-link ( n = 8; red bar) conditions from P15 to 20 (Student's t -test, * P

    Article Snippet: As previously described (Groc et al , ; Heine et al , ), for the x-link experiments, neurons were co-transfected with GluN1-SEP or GluN2B-SEP and Homer 1c-DsRed and incubated with highly concentrated (1:20) polyclonal antibodies directed against GluN1 (Alomone Labs; epitope corresponding to residues 385–399 of the GluN1 subunit), GluN2B (Alomone Labs; same as above), GluN2A (Alomone Labs; same as above) NMDAR subunits or against GFP (Chemicon).

    Techniques: Incubation

    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

    Journal: The EMBO Journal

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

    doi: 10.1002/embj.201386356

    Figure Lengend 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

    Article Snippet: As previously described (Groc et al , ; Heine et al , ), for the x-link experiments, neurons were co-transfected with GluN1-SEP or GluN2B-SEP and Homer 1c-DsRed and incubated with highly concentrated (1:20) polyclonal antibodies directed against GluN1 (Alomone Labs; epitope corresponding to residues 385–399 of the GluN1 subunit), GluN2B (Alomone Labs; same as above), GluN2A (Alomone Labs; same as above) NMDAR subunits or against GFP (Chemicon).

    Techniques: 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

    Journal: The EMBO Journal

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

    doi: 10.1002/embj.201386356

    Figure Lengend 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

    Article Snippet: As previously described (Groc et al , ; Heine et al , ), for the x-link experiments, neurons were co-transfected with GluN1-SEP or GluN2B-SEP and Homer 1c-DsRed and incubated with highly concentrated (1:20) polyclonal antibodies directed against GluN1 (Alomone Labs; epitope corresponding to residues 385–399 of the GluN1 subunit), GluN2B (Alomone Labs; same as above), GluN2A (Alomone Labs; same as above) NMDAR subunits or against GFP (Chemicon).

    Techniques: Diffusion-based Assay, Fluorescence