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Fig. 5. Inhibition of actin dynamics suppresses the effect of activin on spinal morphology. (A) Typical images of synapses of primary cultured hippocampal neurons stained with phalloidin (F-actin, TRITC, red) and anti-synaptophysin (FITC, green). Neurons were treated with activin (100 ng/ml) in the presence or absence of Cyt D (0.1 µM) for 6 hours. Scale bar, 1 µm. (B) Effect of Cyt D on the increase in the average number of synaptophysin-positive puncta per phalloidin-positive punctum induced by activin (100 ng/ml). The y-axis represents the difference from the vehicle control in the average number of presynaptic contacts on each spine. The average number of presynaptic contacts on each spine of control neurons was 1.11. The average of six independent experiments is shown. More than six neurons (two dendrites per neuron) with >350 spines were analyzed for each experiment. Control versus experimental groups: ***P<0.001; ns, P>0.05. Activin versus activin + Cyt D: ***P<0.001. (C) Typical images of primary cultured hippocampal neurons stained with phalloidin (F-actin, TRITC, red), anti-Map2 (FITC, green) and anti-synaptotagmin I (Cy5, blue). Neurons were treated with activin (100 ng/ml) in the presence or absence of Cyt D (0.1 µM) for 6 hours. Scale bar, 10 µm. (D) Cumulative percentages of spine lengths. Neurons were treated with the indicated compounds for 6 hours. Cyt D completely blocked the activin-induced lengthening of dendritic spines. The inset shows the differences in spine length. The average spine length (µm) of control neurons was 1.63. Control versus experimental groups: ***P<0.001; ns, P>0.05. Activin versus activin + CytD: ***P<0.001. Six neurons with >700 spines were examined for each experiment. These experiments were carried out three times and representative data are shown.
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