Figure 3

CC1-EGFP inhibited BDNF-induced pre-synaptic vesicle release in cultured hippocampal neurons. (a) Protocol for FM1-43 labeling and rinsing of pre-synaptic vesicles. Two images were captured before (I) and after (II) BDNF (50 ng/ml, 10 min) treatment. (b,c) CC1-EGFP inhibited BDNF-induced neurotransmitter release. Cultured hippocampal neurons (DIV7) were infected with an EGFP or CC1-EGFP-containing lentivirus. The FM1-43 assay was conducted in the neurons 48 h later according to (a). The releasable FM 1-43 fluorescence intensity (difference between images I and II) was quantitatively measured. Data are shown as the mean±S.E.M. from three independent experiments, >30 neurons per experiment (n=3; *P<0.05, **P<0.01 compared to the vehicle-treated EGFP control group; ^##P<0.01 compared to the BDNF-treated EGFP group). Scale bar, 10 μm. (d) Fluorescence intensity of FM1-43-positive puncta upon K⁺ stimulation. The results showed that CC1-EGFP did not change the number of synaptic vesicles in the active zone. Data of three independent experiments are shown and >30 neurons per experiment. (e–g) CC1-EGFP did not change the density and size of SV2-positive puncta in cultured hippocampal neurons. The numbers of neurons analyzed for each group was >30. Values are shown as the mean±S.E.M. from three independent experiments. Scale bar, 10 μm