The cerebellum slices were transfected by different sets of siRNA on the second day of culture. Old culture medium was replaced by 1 ml of fresh culture medium in each well of the 6-well culture plates before transfection. 100 nM of siRNA was dissolved in 100 ul of 5% glucose solution and mixed with 4 ul of INTERFERin transfection reagent (Polyplus transfection) and incubated for 10 min at room temperature. The mixture was then slowly applied onto the cerebellar slices on top of 1 ml culture medium in three times at 30 min intervals. The resulting concentration of siRNA for transfection would be 10 nM. The transfected cerebellum slices were maintained for 3 days (for P10 culture) or 5 days (for P5 culture) and culture medium was changed every 2–3 days.

Purkinje cells (PCs) are one of the principal neurons in the cerebellar cortex that play a central role in the coordination of fine-tuning body movement and balance. To acquire normal cerebellum function, PCs develop extensive dendritic arbors that establish synaptic connections with the parallel fibers of granule cells to form the proper neuronal circuitry. Therefore, dendritic arborization of PCs is an important developmental step to construct the mature neural network in the cerebellum. However, the genetic control of this process is not fully understood. In this study, Foxp4, a forkhead transcription factor that is expressed specifically in migrating and mature PCs of cerebellum from embryonic stages to adulthood, was knocked down by small interfering RNA (siRNA) in organotypic cerebellar slice culture. When Foxp4 expression was knocked down at postnatal day 5 (P5), no abnormalities for early dendritic remodeling of PCs were observed. However, when Foxp4 was knocked down in P10 cerebellar slices, the organization of PC dendritic arbors was highly impaired, leaving hypoplastic but non-apoptotic cell bodies. The radial alignment of Bergmann glial fibers that associated with PC dendrites was also lost. These results suggest that Foxp4 is dispensable for the early PC dendrite outgrowth, but is essential for the maintenance of PC dendritic arborization and subsequent association with Bergmann glial fibers.