Binding Characteristics of 125i‐labelled Tetanus Toxin to Primary Tissue Cultures From Mouse Embryonic CNS

Abstract— The interaction of 125 I‐labelled tetanus toxin with cells in tissue cultures derived from embryonic CNS has been studied. The optimum toxin binding occurs about 2–3 weeks after transfer of the cells to culture conditions. The amount of label bound per culture was doubled at this time in comparison to the fourth day nfter inoculation. The amount of toxin bound depends on the concentration applied. It reaches its maximum 8 h after application then decreases slowly. Low amounts of radioactivity were still detectable 97 h after washing off the unbound toxin. Up to 80% of the label can be replaced by simultaneous application of‘cold’toxin. Fixation of the toxin is higher at 4°C than at 37°C. Preincubation of the cultures with neuraminidase prevents about 75% of the binding. The presence of cytochalasin B leads to a small but reproducible decrease of binding, whereas colchicine had no measurable effect. The radioactive ( 125 1) material was identified by a double‐isotope technique in disc gel electrophoresis before and after reductive cleavage of its disulphide bonds. In every test is was indistinguishable from 131 I‐labelled toxin added as standard. Our results largely parallel those obtained with synaptosomes and other systems. They suggest that gangliosides might be the acceptor molecules, and that the culture system will be suitable for studying the actions of this toxin in vitro.