Time course and efficiency of protein synthesis inhibition following intracerebral and systemic anisomycin treatment

Since its discovery in the 1960s, anisomycin has been used for studying the impact of protein synthesis for manifold cerebral processes such as long-term plastic changes after learning. The common limitation of nearly all pharmacological experiments, including anisomycin treatment, is to precisely verify the affected brain regions. Here we illustrate anisomycin effects on protein synthesis in distinct brain regions of mice (C57BL/6JOlaHsd), revealing differences between three modes of anisomycin application (subcutaneous, s.c.; intraperitoneal, i.p.; local microinfusions into the hippocampus). Our method is based on inhibition of the incorporation of the radioactively-labelled amino acids [35S]-Methionine/Cysteine into newly synthesised proteins. Washing the brain slices before autoradiography removes pools of amino acids, which have not been incorporated into newly synthesised proteins, thus, illustrating pure protein synthesis. By comparing different routes of systemic anisomycin application (i.p. versus s.c.; 150 mg/kg) it became evident that the effect of i.p. injection of anisomycin is fully reversed after 6 h, whereas s.c. injection is inhibiting protein synthesis in the hippocampus even 9 h after application. Local microinfusions of anisomycin into the hippocampus were shown to have long-lasting effects as well, which reversed as late as 9 h after injection. The diffusion of anisomycin was maximal at 3 h after injection and more precisely confined to the intended area using a lower dose (20 μg/site) instead of the commonly used dose of 62.5 μg/site. The broad time window of anisomycin action, as revealed in our study, has to be considered, if it comes to the interpretation of time course studies within the context of protein synthesis-dependent processes.