Fig. 1. Activin modulates the number of presynaptic contacts made on each postsynaptic spine. (A,B) Cultured primary hippocampal neurons were treated with vehicle (A) or activin (100 ng/ml; B) for 6 hours and then stained with phalloidin to identify F-actin (TRITC, red) and an anti-synaptophysin antibody (FITC, green). Scale bar, 5 µm. The right panels of each figure show higher-magnification images. Scale bar, 1 µm. (C) Quantification of the presynaptic contacts (synaptophysin-positive puncta) on each postsynaptic spine (phalloidin-positive punctum). The average of four independent experiments is shown. Six neurons (two dendrites per neuron) with >250 spines were analyzed for each experiment. ^***P<0.001, control versus activin group, Student's t-test. (D) Populations of phalloidin-positive puncta that are contacted by single or multiple synaptophysin-positive puncta. Dark blue, single contacts; light blue, double contacts; yellow, triple contacts. The average of seven independent experiments is shown. Six neurons (two dendrites per neuron) with >250 spines and ten neurons with >400 spines were analyzed for the control and activin groups, respectively. (E) Activin (100 ng/ml) was added to the culture medium and antibody against activin A (5 µg/ml) was added 2 hours later. The culture was further incubated for 22 hours (24 hours total activin incubation) (left) or 4 days (right). 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 control neurons was 1.09 after 24 hours and 1.14 after 4 days. The average of three independent experiments is shown for the 24-hour incubation, and the total numbers of spines and neurons examined are >900 and 18, respectively. For the 4-day incubation, the numbers of spines and neurons examined are >300 and 6, respectively. Control versus experimental group: ^***P<0.001; ^*P<0.05; ns, P>0.05. Activin group versus activin + antibody group: ^*P<0.05; ^**P<0.01.