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Materials and methods
Materials. 8-Br-cGMP and Rp-8-pCPT-cGMPs were from BIOLOG (Bremen, Germany), NOC-5, LY2109761 Y-27632 and Toxin B (Clostridium difficile) from Calbiochem (Darmstadt, Germany). Nω-nitro-l-arginine methyl ester (l-NAME) and Nω-nitro-d-arginine methyl ester (d-NAME) were from Sigma (Steinheim, Germany). Antibodies against phospho-cofilin (Ser3), cofilin and phospho-PAK4 (Ser474)/PAK5 (Ser602)/PAK6 (Ser560) were purchased from Cell Signaling Technology (Frankfurt, Germany) and β-actin from Sigma.
Animals. Sprague Dawley rats were obtained from Charles River (Sulzfeld, Germany). Animals were maintained in climate- and light-controlled rooms (22 ± 0.5 °C, 12/12 h dark/light cycle) with free access to food and water. All experiments were approved by the local Ethics Committee for research in laboratory animals (RP Darmstadt).
Behavioral experiments have been performed by a axons blinded observer.
Primary neuronal cell culture. For the generation of primary neuronal cell cultures whole fetal brains from 17 day-old embryos were prepared as described previously [17].