Fig. 5: U87 exosomes increase the number of excitatory synapses and decrease inhibitory synapses at DIV 7–12 in hippocampal neurons.

A Representative confocal images of excitatory synapses of hippocampal neurons from control (Ctr) and U87 exosome-treated (Exo) groups at DIV 4 and DIV 7–12. Neurons were identified by β3-tubulin staining (purple channel), and excitatory synapses were identified using double immunostaining with VGLUT1 (green channel)/PSD95 (red channel). Colocalization of pre- and post-synaptic markers is highlighted in yellow and corresponds to bona fide excitatory synapses. C VGLUT1/PSD95 colocalization was evaluated using Mander’s colocalization coefficient (MCC). tM1 corresponds to the fraction of VGLUT1 in compartments containing PSD95, while tM2 to the fraction of PSD95 in compartments containing VGLUT1. B Representative confocal images of inhibitory synapses identified by double immunostaining with VGAT (pre-synaptic, red channel)/GEPHYRIN (post-synaptic, green channel) under the same experimental conditions shown in (A). D VGAT/GEPHYRIN colocalization analysis was obtained as described in (B). Scale bar: 5 μm. Bar graphs show the average ± SEM of four different coverslips prepared from n = 4 distinct preparations. Unpaired Student’s t-test, **p < 0.01. For each coverslip at least 10 dendrites were analyzed. E Upper panels: Representative traces from recordings in voltage-clamp (Black: Control, blue: GASC-479 exosome-treated) showing spontaneous synaptic events evoked by GABA or glutamate (Gray and orange color, respectively). Lower panels: Average peak currents distribution from the recordings at DIV 7–12 for control cells (n = 3) and GASC-479 exosome-treated cells (n = 4) in cultures with low synaptic activity. Left: GABAergic events distribution, Right: Glutamatergic events distribution (*p < 0.05 Kolmogorov–Smirnov test).