Double labeling of neural circuits using horseradish peroxidase and cobalt

This describes for the first time a non-fluorescent method for studying the connectional interrelationship of two neuronal systems in the same histological sections in which the systems are stained in two different colors (blue and brown). Since HRP and cobaltous-lysine are transported in both anterograde and retrograde directions, it is possible to manipulate the experiments to determine, for example, how the terminal fields of two systems overlap in a given neuropil, or cell aggregate, or how the projections of one system relate to retrogradely stained neurons of another system. The simple staining of HRP and cobalt is accomplished on cryostat sections and involves the combined technology of two previously published methods. The results described here are limited to a comparison of the overlap or lack of overlap of inputs to the thalamus from the two eyes in Xenopus, but the method has also proven useful in similar studies on Ambystoma tigrinum and Esox niger.