Fig. 6. The MAP kinase cascade mediates the effects of activin. (A) Cultured primary neurons were stained with anti-phospho-ERK1/2 (pERK1/2, green) at the indicated times after activin treatment (100 ng/ml). Scale bar, 20 µm. (B) Cultured hippocampal neurons and astroglia were treated with tetrodotoxin (1 µM) for 24 hours and then with activin (70, 140 or 280 ng/ml) for 1 hour. Total cellular protein was isolated and subjected to immunoblot analysis. (C) Quantification of immunoblot signals. The ratio of phospho-ERK1/2 signal to total ERK1/2 signal relative to control (without activin) is plotted. ERK1/2 phosphorylation in primary cultured neurons increases after activin treatment but is not altered in activin-treated astroglial-enriched cultures. ^*P<0.005 (one-way ANOVA followed by Fisher's LSD test). (D) Typical images of synapses of primary cultured hippocampal neurons stained with phalloidin (F-actin, TRITC, red) and anti-synaptophysin (FITC, green). The neurons were treated with activin (100 ng/ml) in the presence or absence of the MEK inhibitor U0126 (1 µM) for 6 hours. Scale bar, 1 µm. (E) Effect of U0126 (1 µM) on the increase in average number of synaptophysin-positive puncta per phalloidin-positive puncta 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 in controls was 1.03. The average of five independent experiments is shown. 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 + U0126: ^***P<0.001. (F) Effect of U0126 (1 µM) on spine lengthening induced by activin (100 ng/ml). The activin-induced lengthening of spines was blocked by U0126. The inset shows the average spine length. The average spine length (µm) of control neurons was 1.71. Control versus experimental groups: ^***P<0.001; ns, P>0.05. Activin versus activin + U0126: ^***P<0.001. Six neurons with >600 spines were examined for each experiment. These experiments were carried out four times and representative data are shown.