Figure 1. ADF is present in pre- and postsynaptic structures of excitatory synapses.

(A) Immunoblot analyses demonstrated the specificity of the ADF antibody used for expression and localization studies: no band was detectable in cortical (CX), hippocampal (HIP) or striatal (STR) protein lysates from homozygous ADF mutants (−/−). N-cofilin levels were unchanged in lysates from homozygous ADF mutants. (B) Immunoblot analysis demonstrated the broad expression of ADF in the brain throughout postnatal development (postnatal day 0 (P0) to P80). CB: cerebellum, BS: brainstem. (C) Immunocytochemistry of cultured hippocampal pyramidal cells after 21 days in culture demonstrating the presence of ADF (green) in the cell body and in neurites. F-actin-rich structures were visualized with fluorescent-labeled phalloidin (red). Scale bar: 50 µm. (D) High magnification of a dendritc shaft, demonstrating the presence of ADF in phalloidin-positive, F-actin-rich structures such as dendritic spines. Scale bar: 5 µm. (E) Co-labeling with synaptophysin (synapto; red) revealed the presence of ADF (green) in presynaptic structures. Scale bar: 5 µm. (F) Likewise, ADF (green) co-localized with PSD-95 (red), a marker of the postsynaptic density. Scale bar: 5 µm. Small images in D–F: binary masks (green or red channel) and merged masks of dendritic structure indicated by dashed boxes. (G+H) Representative electron micrographs of the CA1 and CA3 stratum radiatum demonstrate localization of ADF in presynaptic boutons (b) and dendritic spines (sp). Arrows point to exemplarily gold particles that label ADF. Scale bars: 150 nm in G, 200 nm in H. (I) In the CA1 and CA3 region, density of gold particles was significantly higher in presynaptic boutons than in dendritic spines.