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First published online 16 October 2007
doi: 10.1242/jcs.012450

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Activin increases the number of synaptic contacts and the length of dendritic spine necks by modulating spinal actin dynamics

Yoko Shoji-Kasai¹, Hiroshi Ageta¹, Yoshihisa Hasegawa², Kunihiro Tsuchida^3,*, Hiromu Sugino^3, and Kaoru Inokuchi^1,4,5,

¹ Mitsubishi Kagaku Institute of Life Sciences, MITILS, 11 Minamiooya, Machida, Tokyo 194-8511, Japan
² Laboratory Animal Science, Kitasato University School of Veterinary Medicine and Animal Sciences, Towada, Aomori 034-8628, Japan
³ Institute for Enzyme Research, University of Tokushima, Tokushima 770-8503, Japan
⁴ Graduate School of Environment and Information Sciences, Yokohama National University, Yokohama 240-8501, Japan
⁵ Japan Science and Technology Agency, CREST, Kawaguchi, Saitama 332-0012, Japan

View larger version (42K): [in this window] [in a new window]   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.

View larger version (49K): [in this window] [in a new window]   Fig. 2. Live imaging of a cultured neuron reveals spine lengthening after activin treatment. (A,B) Fluorescence images of dendrites of a cultured hippocampal neuron. pEGFP-transfected neurons were treated with activin (100 ng/ml; B) or vehicle (A) and observed for 6 hours. Upper and lower panels are before and after treatment, respectively. Arrowheads indicate spines whose shape changed by 6 hours. Scale bar, 10 µm. (C,D) Length change of individual spines before and after treatment; 38 control spines were treated with BSA (C), and 40 spines were treated with activin (D).

View larger version (51K): [in this window] [in a new window]   Fig. 3. Activin modulates spinal morphology. (A,B) Cultured primary hippocampal neurons were treated with activin (100 ng/ml) (B) or vehicle (A) for 6 hours and stained with phalloidin (for F-actin staining, TRITC, red), anti-Map2 (FITC, green) and anti-synaptotagmin I (Cy5, blue). Left, low magnification; right, high magnification. Scale bars, 10 µm. (C-I) Quantification of changes in spine F-actin morphology after activin treatment (100 ng/ml, except D). The cumulative percentages of spine length (C,D,E,H) and area (G), the average change in spine length (insets in C,D,E,H), the average spine length (I), the average spine area (inset in G), and the average spine density (F) are shown. Spine lengthening was observed 2 hours after activin treatment commenced and spine length increased over 6 hours (C, inset). Increases in spine length were activin-dose dependent (D, inset). Spine size (G) and spine density (F) did not change significantly after activin treatment. (E) Follistatin (250 ng/ml) inhibited the effect of activin, which reveals its specificity. (H) Tetrodotoxin (TTX; 1 µM or 2 µM) did not block the activin effect. The average spine length (µm) in control neurons was 1.58 (C), 1.86 (D), 1.71 (E) and 1.41 (H). Control versus experimental groups: *P<0.05; **P<0.01; ***P<0.001; ns, P>0.05. The total numbers of spines and neurons, respectively, that were examined in each experiment are: >300 and >4 (C), >400 and >5 (G), >1200 and >16 (D), >1300 and 10 (E), 600 and 6 (H), and400 and >6 (I). More than 15 dendrites were examined in F. These experiments were carried out twice (C,D), five times (E,G), three times (F,H) and seven times (I), and representative data are shown.

View larger version (26K): [in this window] [in a new window]   Fig. 4. The effect of activin on spine morphology does not require de novo protein synthesis or RNA synthesis. (A) The effect of cycloheximide on spine lengthening induced by activin (100 ng/ml). Cultured hippocampal neurons were treated with the indicated compounds for 6 hours. (B) The protein-synthesis inhibitors cycloheximide (3 µM) and emetine (0.1 µM), and the RNA-synthesis inhibitor actinomycin D (1 µM), did not have a significant effect on average spine length. Cultured hippocampal neurons were treated with the indicated compounds for 6 hours. The average spine length (µm) of control neurons was 1.49 (cycloheximide), 1.58 (emetine) and 1.58 (actinomycin D). Control versus experimental groups: *P<0.05; **P<0.01. The total numbers of spines and neurons that were measured in each experiment were: >700 and 8 (cycloheximide), >600 and 6 (emetine), and >500 and 6 (actinomycin), respectively. These experiments were carried out five times (cycloheximide), three times (emetine) and twice (actinomycin), and representative data sets are shown.

View larger version (43K): [in this window] [in a new window]   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.

View larger version (33K): [in this window] [in a new window]   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.