A reliable method for culture of dissociated mouse cerebellar cells enriched for Purkinje neurons

The cerebellar Purkinje neuron (PN) serves as an important model in studies of neuronal development in the mammalian central nervous system. Dissociat ed PN preparations maintained in an in-vitro environment with simplified ce llular and biochemical conditions can facilitate molecular analyses of neur onal development. Here we describe a reliable method to prepare dissociated cultures of mouse cerebellar neurons maintained with a serum-free, Dulbecc o's modified Eagle's medium/F-12 nutrient-based medium, which facilitates P N survival and dendritic differentiation. The survival of mouse PNs in this culture was maximized when cerebellar cells were (1) taken from prenatal a nimals, (2) dissociated with papain, and (3) seeded at a density of 5 000 0 00 cells/ml or higher. Dissociated PNs prepared by our method from mice of embryonic day 18 (E 18) reproduced several morphological and electrophysiol ogical changes seen in intact postnatal rodents with similar time-courses. Therefore, our culture method offers a useful model for investigating molec ular mechanisms underlying postnatal neuronal development. (C) 2000 Elsevie r Science B.V. All rights reserved.