glun1  (Alomone Labs)

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

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

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

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

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

Journal: Scientific Reports

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

doi: 10.1038/s41598-017-08695-3

Figure Lengend 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

Article Snippet: As previously described for the x-link experiments , neurons were incubated with highly concentrated (1:10) polyclonal antibodies directed against GluN1 (Alomone Labs; epitope corresponding to residues 385–399 of the GluN1 subunit).

Techniques: Diffusion-based Assay

Journal: Scientific Reports

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

doi: 10.1038/s41598-017-08695-3

Figure Lengend 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

Article Snippet: As previously described for the x-link experiments , neurons were incubated with highly concentrated (1:10) polyclonal antibodies directed against GluN1 (Alomone Labs; epitope corresponding to residues 385–399 of the GluN1 subunit).

Techniques:

Journal: Frontiers in Neuroanatomy

Article Title: Phenotype and Distribution of Immature Neurons in the Human Cerebral Cortex Layer II

doi: 10.3389/fnana.2022.851432

Figure Lengend Snippet: Expression of NMDA receptors (GluN1) and apposed puncta expressing VGLUT1 and GAD67 on DCX immunoreactive cells. (A) Confocal microphotographs of the temporal cortex showing the expression of GluN1 in small type I (A1) and larger type I DCX + cells (A2) . (B) DCX immunoreactive type II cell (green) in the occipital cortex layer II showing puncta expressing the excitatory marker VGLUT1 (red) apposed to its soma and dendrite (arrows). (C) DCX immunoreactive type II cell (green) in the temporal cortex layer II. Note the presence of GAD67 expressing puncta (red) apposed to its soma and dendrite (arrows). (A1) Is a single confocal plane, (A2,B,C) are 2D projections of 4 (A2) 9 (B) and 12 (C) consecutive confocal stacks (0.38 μm apart). Scale bar: 10 μm for (A,B4,B5,C4,C5) ; 40 μm for (B1–3,C1–3) . All confocal images in this figure were from neurosurgical samples.

Article Snippet: Pre-absorption of GluN1 antibody with immunogen peptide (Alomone labs) eliminates all immunostaining (manufacturer’s and own results in our tissue).

Techniques: Expressing, Marker

Journal: The Journal of Experimental Medicine

Article Title: Lupus antibodies induce behavioral changes mediated by microglia and blocked by ACE inhibitors

doi: 10.1084/jem.20180776

Figure Lengend Snippet: HMGB1 from neurons and microglia targets neuron-derived C1q to synapses. (A) Neurons were cultured alone and stained for GluN1 (green), labeled HMGB1 (red), MAP2 (cyan), and DAPI (blue). (B) Left, neurons were stimulated with increasing concentrations of NMDA and stained for HMGB1 (red), PSD-95 (green), and MAP2 (cyan). Right, colocalization of HMGB1 and PSD-95 shows a significant dose effect ( n = 10 quantified neurons; ***, P

Article Snippet: Anti-MAP2 antibody (Sigma-Aldrich), anti-postsynaptic density protein 95 (PSD-95) antibody (Millipore), anti-NMDAR subunit 1 extracellular (GluN1) antibody (Alomone Labs), antiHMGB1 antibody (Novus), and anti-C1q antibody (Abcam) were used for immunofluorescence staining.

Techniques: Derivative Assay, Cell Culture, Staining, Labeling