Light and electron microscopical visualization of anterogradely labelled corticospinal growth cones using a new combination of HRP staining techniques.

Up until now, the ultrastructural visualization of growth cones of developing long fibre tracts could only be achieved by horseradish peroxidase (HRP) application 'en route', resulting in axonal damage, which in turn may affect growth cone morphology. Besides, this technique results in labelling of passing fibres, thus hampering the identification of axon origin as well as the interpretation of growth cone configuration. In the present investigation a new combination of HRP staining and intensification techniques is presented which makes it possible to visualize anterogradely labelled corticospinal growth cones over long distances in developing rat spinal cord at the light as well as the electron microscopical level. HRP was applied to the originating cells of the corticospinal tract, located in the sensorimotor cortex, and after 24 h was visualized using a procedure which essentially consists of 3 subsequent steps: first a tetramethylbenzidine (TMB)/ammoniumheptamolybdate (AHM) reaction; second diaminobenzidine (DAB)/nickel (Ni) stabilization and finally glucose oxidase intensification. As was verified at the EM level, the staining procedure here described reveals a complete intense black staining of HRP-labelled growth cones of outgrowing corticospinal axons. Therefore, the method described here guarantees a correct analysis of growth cone morphology at the light microscopical and the ultrastructural level. The present procedure is especially valuable in studying the development of long central nervous fibre systems.