Sequential double labelling with different fluorescent dyes coupled to dextran amines as a tool to estimate the accuracy of tracer application and of regeneration

Abstract

We present a technique to estimate the accuracy of a given application procedure for neuronal tracers. In a second series of animals we used this technique for the estimation of successful regeneration of peripheral nerves. Dextran amine coupled to rhodamine was applied to the cut trochlar nerve in Xenopus tadpoles. To assess the accuracy of tracer application, experiments were done in which a second dye, dextran amine coupled to fluorescein, was applied after 1 day proximal to the first dye. More then 90% of all trochlear motoneurons were doubly labelled after this procedure. Their total numbers were not significantly different from numbers obtained after single labelling with HRP in a comparable age group. To assess success of regeneration after 5 and 8 days, the second application of fluorescein dextran amine was distal to the first application side. Statistically significant differences suggest incomplete regeneration of many neurons. After 42 days the numbers of singly and doubly labelled motoneurons was in the same proportion as before regeneration. This suggests that about 90% of the surviving motoneurons had successfully regenerated back to the periphery.