Fig 2: Comparison of widefield microscopy methods in the imaging of tissue sections. Mouse brain sections (60 µm thick) immunofluorescently labeled with antibodies against Homer1 (Homer; red in B and C), Shank2/ProSAP1 (Shank2; green) and MAP2 (white) imaged by 3D-Zoom microscopy (A), a slide scanning microscope (B) and a conventional widefield microscope (C), respectively. DNA was counter-stained with DAPI (blue). Note that fluorescence signals of Homer staining in (A) were not captured due to lack of an appropriate fluorescence excitation. Instead, an additional place holder fluorescence channel was captured (ND, red) to allow later comparison of acquisition speed with the other widefield methods (see Table 1). (A2) Magnified view of a cortical area boxed in (A1). Individual immunofluorescence signals of this subregion are shown in monochrome in A3 to A6. An arrow in A3 depicts an example of a cell nucleus. (B1) The same sample as in A imaged in 3D using a multi-slide scanning microscope. (B2) Single optical section shown as a magnified view of the subregion boxed in B1. (B3 to B6) Individual immunofluorescence channels of the image in B2 in monochrome. Arrows in B3 and B4 mark individual chromo-centers within single nuclei and MAP2-positive dendrites, respectively, and thereby highlight an improved resolution provided by the multi-slide scanning microscope. (C1) The same sample imaged in 3D using a conventional widefield microscope. (C2) A single optical section was selected from the dataset shown in C1 (marked with a white box) and split into individual fluorescence channels (C3 to C6). Images C2d to C6d show the same subregion after deconvolution. Individual chromo-centers within single nuclei as well as MAP2-positive dendrites become clearly resolvable after deconvolution (Arrows in C3d and C4d, respectively).

Fig 3: 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.

Fig 4: Generation and basic characterization of Pv-Cre;Shank2fl/fl mice. (A) Breeding scheme for the production of WT and Pv-Cre;Shank2fl/fl conditional KO (cKO) mice. Pv-Cre indicates mice heterozygous for the modified Pv allele, Pv-IRES-Cre; Pv-wild indicates normal WT mice without a Cre gene insert. (B) Genotyping of WT and Pv-Cre;Shank2fl/fl mice, by PCRs on WT and cKO cerebellar samples (4 months). Note that only Pv-Cre;Shank2fl/fl mice are positive for both the Pv-Cre allele and the Shank2 KO allele. (C) Levels of Shank2 protein in different brain regions of WT and Pv-Cre;Shank2fl/fl mice (4 months). Ctx, cortex; Str, striatum; Hp, hippocampus; Cb, cerebellum. (n = 4 mice for WT and cKO, *P < 0.05, ns, not significant, Mann-Whitney U test). (D) Pv-Cre;Shank2fl/fl mice (16 weeks) show normal numbers of PV-positive neurons and normal intensities of PV protein signals, as shown by the ratio of PV-positive cells and NeuN (neuronal marker)-positive cells and the intensity of PV protein signals normalized to cell area in Cg1 and M2 cortical regions. Note that a 200 × 200 µm block (middle) rather than the whole area (800 × 1000 µm; left) is shown to better visualize individual cells (n = 8 mice for WT and 7 mice for cKO, ns, not significant, Student’s t-test) The images from the Allen Mouse Brain Atlas was used in (D) (brain diagrams on the left; Lein et al., 2007). Scale bar, 50 µm.

Fig 5: Shank2 protein levels are decreased in the thalamic reticular nucleus (TRN) and cerebellum in Pv-Cre;Shank2fl/fl mice. (A) Decreased Shank2 protein levels in the TRN and cerebellum of Pv-Cre;Shank2fl/fl mice (4 months), revealed by immunofluorescence staining of sagittal brain sections. Cb, cerebellum; TRN, thalamic reticular nucleus. Scale bar, 1 mm. (B) Reduced punctate Shank2 signals in dendrites and cell body regions around PV-positive cells in the TRN and cerebellum of Pv-Cre;Shank2fl/fl mice. Scale bar, 20 µm.