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