Formation of the corticopontine projection in a long-term culture system

The reestablishment in vitro of the corticopontine projection was studied in organotypic co-cultures of cortex and pens of rats 0 (day of birth) - 3 days old. After 2-3 weeks in vitro, application of the lipophilic tracer DiI in the pontine explant retrogradely stained in layer V of the cortical explant pyramidal neurons which were characterized by large somata and spiny dendrites with an apical dendrite that reached upper cortical layers II/III. The projection developed only in co-cultures from rats 0-2 days old when the pontine explant was placed in close vicinity either to the white matter or the pial surface of the cortical explant. Control experiments demonstrated the specificity of the corticopontine projection in vitro by showing that the projections of the layer V pyramidal cells to the pens did not reflect a non-directed outgrowth pattern with subsequent survival of axons contacting the target explant. Our findings demonstrate that morphology and laminar position of corticopontine projection neurons in vitro are similar to those in vivo and thus support the "organotypic" nature of the explant co-culture system.