Demonstration of connections between cortex and superior colliculus in vitro by the use of biocytin and HRP

To study the morphology of neurons in the superior colliculus (SC) and visual cortex (VC) in vitro, the retro- and anterograde tracers biocytin and horseradish-peroxidase (HRP) were applied in cortical and tectal explant co-cultures. Injection of biocytin alone into the SC explant after up to 12 days in vitro revealed the detailed morphology of retrogradely labelled SC cells, comparable to cell types previously described in situ. However, only in 2 co-cultures out of 110 did injections of biocytin alone into the SC result in retrograde labelling of cortical cells; the label was very weak and did not allow detailed analysis of the morphological features. Only with a combination of biocytin and HRP, were the pyramidal cells of layer V adequately stained. Our result show that: 1. It is possible to use actively transported neuronal tracers in cultures, 2. anterograde as well as retrograde labelling reveals intrinsic connections in an SC explant, 3. connections between the two explants (both cortico-tectal as well as tecto-cortical) could be labelled, and 4. a combination of biocytin and HRP resulted in a much better retrograde labelling and thereby demonstration of cellular morphology of layer V pyramidal cells in VC than biocytin alone.