Epigenetic factors controlling the development of avian Purkinje neurons

Using a unique protocol, we have developed an avian neuron culture system in which a high yield of Purkinje neurons is obtained more readily than with pre-existing methods. Purkinje neurons were identified in vitro using the specific antibodies calbindin and cyclic GMP-kinase. Survival of Purkinje neurons was dependent on astrocyte contact and enhanced by astrocytic factors supplied to the medium by a monolayer of astrocytes grown on coated membranes suspended in the culture wells but not in contact with the neurons. The age of the cerebellum from which astrocytes were obtained was shown to affect the morphological development of the Purkinje neurons suggesting the developmentally-regulated expression of growth factors. However, in the presence of the astrocytes, Purkinje neurons could only progress to a limited stage of development based on morphological criteria. The addition to the culture of cerebellar granule neurons at a time of Purkinje neuron development that they would expect to encounter them in vivo resulted in a shift of Purkinje neurons to a mature phenotype. This maturation effect was increased in response to increasing levels of granule neurons, but was independent of the granule neuron ages used. This system offers significant advantages over other Purkinje neuron culture systems and will be useful for studying the extrinsic factors involved in Purkinje neuron development and histogenesis.