Pilocarpine-induced epilepsy is associated with actin cytoskeleton reorganization in the mossy fiber-CA3 synapses

• F-actin density is decreased in the hippocampus of mice that underwent the pilocarpine status epilepticus.
• The F-actin changes were consistent with postsynaptic densities in the CA3 region.
• F-actin puncta were more highly clustered and enlarged in the epileptic mice.
• Synapsin I was not altered in the CA3 region of epileptic mice.
• Cytoskeletal changes may be associated with functional changes related to hippocampus sclerosis.

SummaryDramatic structural changes have been demonstrated in the mossy fiber-CA3 synapses in the post status epilepticus (SE) animals, suggesting a potential reorganization of filamentous actin (F-actin) network occurring in the hippocampus. However, until now the long-term effects of SE on the synaptic F-actin have still not been reported. In this study, phalloidin labeling combined with confocal microscopy and protein analyses were adopted to investigate the effects of pilocarpine treatment on the F-actin in the C57BL/6 mice. As compared to the controls, there was ∼ 43% reduction in F-actin density in the post SE mice. Quantitative analysis showed that the labeling density and the puncta number were significantly decreased after pilocarpine treatment (p < 0.01, n = 5 mice per group, Student's t-test). The puncta of F-actin in the post SE group tended to be highly clustered, while those in the controls were generally distributed evenly. The mean puncta size of F-actin puncta was 0.73 ± 0.19 μm2 (n = 1102 puncta from 5 SE mice) in the experimental group, significantly larger than that in the controls (0.51 ± 0.10 μm2, n = 1983 puncta from 5 aged-matched control mice, p < 0.01, Student's t-test). These observations were well consistent with the alterations of postsynaptic densities in the same region, revealed by immunostaining of PSD95, suggesting the reorganization of F-actin occurred mainly postsynaptically. Our results are indicative of important cytoskeletal changes in the mossy fiber-CA3 synapses after pilocarpine treatment, which may contribute to the excessive excitatory output in the hippocampal trisynaptic circuit.