Functional and reliable in vitro models of Parkinson's disease (PD) are valuable for studying mechanisms of dopaminergic degeneration before proceeding to animal testing. At present, all in vitro models involve substitute cell types and thus their direct relevance to PD is questionable. Here, we describe an organotypic culture model which conserves the 3D architecture of the nigro-striatal pathway, together with the subventricular zone and cerebral cortex, and recapitulates a specific pattern of dopaminergic degeneration which is the principal hallmark of PD. The organotypic culture is kept in vitro for up to 12 days and dopaminergic degeneration is induced by the simple cutting of dopaminergic fibers. This organotypic model represents a rapid and useful method (30 min/pup for preparation and up to 12 days of cultivation) to investigate in vitro the mechanisms underlying neuronal death and protection, as well as neurogenesis and repair after nigro-striatal neurodegeneration.