Observation of the highly organized development of granule cells in rat cerebellar organotypic cultures.

Cerebellar slices of 9-day-old rats were cultured for a week at the interface between air and a culture medium containing horse serum and hormone cocktail, and the development of granule cells was characterized morphologically. The typical layered structure of the cerebellar cortex was well preserved during the cultivation. Many granule cells in the external granular layer (EGL) proliferated actively within the early culture period. They were migrating downward at 3 days in vitro (DIV) and almost completed the migration to the internal granular layer (IGL) after 6 DIV. In the middle and upper molecular layer (ML), parallel fibers were elongated horizontally, which is the orientation 90 degrees rotated compared to that in vivo. They formed synapses with Purkinje cell dendrites. Regional differences in synapse density and maturity existed which might reflect a gradient in progressive synapse formation comparable to that in vivo. Thus, a serial process of highly organized development of granule cells was realized for the first time in vitro, although some spatial or temporal modifications existed. Such a culture system could be a useful experimental model for the study of cellular and molecular mechanisms of spatiotemporally organized neuronal development.