Journal: Scientific Reports

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

doi: 10.1038/s41598-020-69454-5

Figure Lengend 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

Article Snippet: Single-particle tracking For endogenous GluN2A or 2B quantum dot (QD) fluorescence particle tracking, hippocampal neurons were incubated with an antibody against the N-terminal extracellular domain of GluN2A (ACG-002 1:500, Alomone Labs) or GluN2B subunits (ACG-003 1:500, Alomone Labs) for 10 min. Neurons were then washed and incubated for 5 min with QD 655 anti-rabbit IgG (Q-11421MP, Invitrogen ).

Techniques: Marker

Journal: Scientific Reports

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

doi: 10.1038/s41598-020-69454-5

Figure Lengend 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

Article Snippet: Single-particle tracking For endogenous GluN2A or 2B quantum dot (QD) fluorescence particle tracking, hippocampal neurons were incubated with an antibody against the N-terminal extracellular domain of GluN2A (ACG-002 1:500, Alomone Labs) or GluN2B subunits (ACG-003 1:500, Alomone Labs) for 10 min. Neurons were then washed and incubated for 5 min with QD 655 anti-rabbit IgG (Q-11421MP, Invitrogen ).

Techniques: Diffusion-based Assay