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. 2022 Mar 3;145(8):2849–2868. doi: 10.1093/brain/awac083

Figure 2.

Figure 2

Large Aβ-EVs alter dendritic spine morphology and synaptic plasticity in cultured neurons. (A) Schematic representation of EV delivery by optical tweezers to RFP-expressing dendrite, preceded and followed by time-lapse imaging of RFP-positive dendritic spines. A z-stack of RFP-positive dendrites was first acquired with a spinning disk microscope, then a low amount of EVs was added to the cell medium and one EV was captured (trapped) above the neurons by the infrared laser tweezers and placed in contact with the imaged dendrite (bright field). After 30 s the laser was switched off, EV adhesion was checked and confocal images were collected at the indicated time points. (B) Representative confocal images taken before and 30 min after contact of ctrl-EVs (centre) or Aβ-EVs (right) following the procedure described in A, showing dendritic spine changes in proximity (top) and far from the EV contact site (bottom). Red and orange circles indicate the site of EV contact. White arrows point to newly generated protrusions. Red arrows point to enlarged spines. Orange arrows point to thinned spines. On the left, dendritic spine images at 0 and 30 min after vehicle addition. Scale bar: 10 µm. (C and D) Temporal analysis of dendritic spine density around the contact site (<7 µm, C) and far from the contact site (>60 µm, D; n = 6 dendrites/condition, 12 experiments). Values are normalized to the pre-adhesion condition (two-way repeated measures ANOVA, followed by the Holm–Sidak method; close to the contact site: P = 0.013 ctrl-EVs versus vehicle; P < 0.001 Aβ-EVs 30 and 40 min versus 0; far from the contact site: P = 0.937). (E–H) Temporal analysis of the density of immature (thin) and mature (mushroom and stubby) dendritic spines around the contact site (E and G) and far from the contact site (F and H) after adhesion of Aβ-EVs or ctrl-EVs or in vehicle-treated neurons (immature spines at the contact site: P < 0.01 Aβ-EVs versus ctrl-EVs and versus vehicle; P < 0.001 Aβ-EVs 20, 30, 40 min versus 0; immature spines far from the contact site: P = 0.656; mature spines at the contact site: P < 0.001 Aβ-EVs versus ctrl-EVs; P = 0.015 Aβ-EVs versus vehicle; P < 0.01 ctrl-EVs versus vehicle; P < 0.001 ctrl-EVs 20, 30, 40 min versus 0; mature spines far from the contact site, ns). (I) Representative images showing Shank-2/Bassoon double-positive puncta in vehicle-treated neurons, neurons exposed to ctrl-EVs or Aβ-EVs. Scale bar: 1 µm. (J) The box plot shows the corresponding fraction of juxtaposed pre- and post-synaptic puncta relative to Bassoon positive synaptic puncta (Kruskal–Wallis one-way ANOVA on Ranks, followed by Dunn’s method, P < 0.05 Aβ-EVs versus vehicle; n = 3 experiments). Box plot shows the median (central line) and mean (X), upper and lower quartile (box limits), maximum and minimum values (whiskers). (K) Representative traces of mEPSCs recorded from control neurons (vehicle) and neurons exposed to Aβ-EVs or ctrl-EVs for 1 h, before and after induction of synaptic plasticity. Vertical scale bar: 5 pA; horizontal scale bar: 1 s. (L) Temporal plot of mEPSC frequency changes showing that glycine (Gly, 200 µM 3 min in 0 Mg++, preceded by 1 min 0 Mg++) induced a long-lasting increase in mEPSC frequency in both vehicle- and ctrl-EV-treated neurons but not in neurons exposed to Aβ-EVs for 1 h (two-way repeated measures ANOVA, followed by the Holm–Sidak method; 2.931 ± 0.808 ‘vehicle’ fold change from baseline, P = 0.002; 2.409 ± 0.549 ‘ctrl-EVs’ fold change from baseline, P = 0.027; 0.942 ± 0.156 ‘Aβ-EVs’ fold change from baseline, P = 0.902; vehicle versus Aβ-EVs, P = 0.012; ctrl-EVs versus Aβ-EVs post Gly, P = 0.009; vehicle, n = 6 cells; ctrl-EVs, n = 5; Aβ-EVs, n = 8; 7 experiments). Data are expressed as mean ± SEM.