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AM251 treatment has no effect on synaptic activity, cell viability and CB1 receptor expression. A Representative electrophysiological traces and group data of average mEPSC amplitude and frequency of control (n = 10) and AM 251 treated (n = 9) cells (scale bar: 20 pA, 200 ms). B Fluorescence images of Hoechst and propidium iodide double staining in cortical neurons and summary bar graph showing lack of cytotoxic effects after AM251 treatment (scale bar: 40 μm). C, D Confocal microscopy images and summary bar graph showing expression of CB1 receptors in control and AM251 treated cultured neurons (scale bar: 20 μm). All data are expressed as mean ± standard error of the mean. CB1 type 1 cannabinoid receptor, mEPSC miniature excitatory post-synaptic current. (PPT 1143 kb)

AM251 treatment has no effect on synaptic activity, cell viability and CB1 receptor expression. A Representative electrophysiological traces and group data of average mEPSC amplitude and frequency of control (n = 10) and AM 251 treated (n = 9) cells (scale bar: 20 pA, 200 ms). B Fluorescence images of Hoechst and propidium iodide double staining in cortical neurons and summary bar graph showing lack of cytotoxic effects after AM251 treatment (scale bar: 40 μm). C, D Confocal microscopy images and summary bar graph showing expression of CB1 receptors in control and AM251 treated cultured neurons (scale bar: 20 μm). All data are expressed as mean ± standard error of the mean. CB1 type 1 cannabinoid receptor, mEPSC miniature excitatory post-synaptic current. (PPT 1143 kb)

Botulinum toxins BoNT/A and BoNT/E, which inhibit neurotransmitter release by cleaving SNAP-25, have been reported to block synaptic vesicle recycling preferentially at excitatory but to much lesser degree at inhibitory synapses (Verderio et al., 2004). This evidence has recently been complemented by the observation that BoNT/A or BoNT/E affect predominantly excitatory with respect to inhibitory neurotransmitter release from rat synaptosomes (Verderio et al., 2007). To prove that resistance of GABAergic terminals did not depend on differences in toxin receptors, the traffic of the toxins was examined in cultured neurons. The results indicated that BoNT/A and BoNT/E are able to equally enter and translocate into the cytosol in both types of neurons. Interestingly, exogenous expression of SNAP-25 in GABAergic neurons conferred sensitivity to BoNT/A. These results are opened to different interpretations: first, SNAP-25 might substitute a putative, endogenous BoNT-resistant SNARE; second, the 1–197 fragment may act as dominant negative for exocytosis. Also, it has been shown that SNAP-25 negatively modulates calcium responsiveness to depolarization (Pozzi et al., 2008) and that BoNT/A-inhibition can be overcome by high Ca 2+ concentrations (Capogna et al., 1997). A third possibility, therefore, is that exogenous expression of SNAP-25, by reducing Ca 2+ responsiveness of GABAergic neurons, may confer sensitivity to BoNT/A. These possibilities are currently under evaluation.

β-Amyloid is one of the main pathological hallmarks of Alzheimer’s disease and plays a major role in synaptic dysfunction. It has been demonstrated that β-amyloid can elicit aberrant excitatory activity in cortical-hippocampal networks, which is associated with behavioral abnormalities. However, the mechanism of the spreading of β-amyloid action within a specific circuitry has not been elucidated yet. We have previously demonstrated that the motion of microglia-derived large extracellular vesicles carrying β-amyloid, at the neuronal surface, is crucial for the initiation and propagation of synaptic dysfunction along the entorhinal-hippocampal circuit. Here, using chronic EEG recordings, we show that a single injection of extracellular vesicles carrying β-amyloid into the mouse entorhinal cortex could trigger alterations in the cortical and hippocampal activity that are reminiscent of those found in Alzheimer’s disease mouse models and human patients. The development of EEG abnormalities was associated with progressive memory impairment as assessed by an associative (object-place context recognition) and non-associative (object recognition) task. Importantly, when the motility of extracellular vesicles, carrying β-amyloid, was inhibited, the effect on network stability and memory function was significantly reduced. Our model proposes a new biological mechanism based on the extracellular vesicles mediated progression of β-amyloid pathology and offers the opportunity to test pharmacological treatments targeting the early stages of Alzheimer’s disease.