Double labelling of retinofugal projections in the cat: a study using anterograde transport of 3H-proline and horseradish peroxidase.

A new method is described for combining 3H-proline and horseradish peroxidase (HRP) as anterograde neuronal tracers. By this method the presence of both substances can be demonstrated in the same histological section. We developed this method to investigate the retinofugal projections from the two eyes in the cat. One eye was injected with 3H-proline the other with HRP. Cryostat sections of the brain were mounted on emulsion coated slides in the dark. Sections were first exposed to the emulsion for 2-3 weeks at -40 degrees C and developed for autoradiography. Only then they were reacted for HRP-activity with tetramethylbenzidine (TMB). Keeping to this sequence autoradiographic procedures could not abolish the HRP-reaction product and silver grains and TMB can be visualized on the same slide. The wellknown projection pattern in the lateral geniculate nuclei was confirmed as a control for the new method. In the superior colliculus and in the pretectum a clear overlap of retinal terminals from the two eyes could be demonstrated for the first time.