Fig. 5
The release method detects alterations of EV biogenesis and isolates intact EVs that propagate in the brain. a Schematic representation of the experiment of inhibition or activation of exosome biogenesis/secretion by GW4869, an inhibitor of neutral sphingomyelinase, and Picrotoxin (PTX), an inhibitor of GABAA receptors that increases excitatory neuronal firing. b–d Brain tissue was incubated with GW4869 or PTX. Nanoparticle tracking analysis (NTA) of the number of particles (b), particles divided by size (c), and relative abundance of smaller (50-150nm) and bigger (>150nm) EVs (d) (Nvehicle = 5, NGW4869 = 3 NPTX = 3). e Schematic representation of the stress protocol timeline. f Body weight change as a measurement of stress efficacy. g Percentage of freezing behavior in the Contextual fear conditioning test. h Percentage of immobility in the Tail suspension test. i Nanoparticle tracking analysis (NTA) of the number of particles by tissue weight detected in stressed and control animals. j microBCA analysis of the amount of protein by tissue weight in stressed and control animals (k) (behaviour: Ncontrol = 10, Nstress = 10; EVs: Ncontrol = 4, Nstress = 4). k Schematic representation of the DiI-labeled EVs injected into the outer molecular layer (OML) of the dentate gyrus of mouse hippocampus and their spreading to the granule cell layer (GCL). l Confocal images showing Dil-labeled EVs (red) at the site of injection (OML) at 1-day post-injection and the spread of Dil signal onto the GCL of dentate gyrus 4 weeks later (N1day = 8, N4weeks = 8). All data shown represent mean ± SEM *p < 0.05 by two-tailed Welch’s t-test or one-way ANOVA