Maintenance of immunocytologically identified Purkinje cells from mouse cerebellum in monolayer culture

Abstract

Listen

Translate article

Purkinje cells were identified in monolayer cultures obtained from trypsin-dissociated cerebella of embryonic and early postnatal mice by the Purkinje cell-specific monoclonal antibodies PC1, PC2, PC3 and UCHT1. These cells also expressed the neuronal marker L1 antigen but not the glial markers, glial fibrillary acidic protein or 04 antigen. They also expressed tetanus toxin receptors, PC4, M1 and Thy-1 antigens. Survival of Purkinje cells was best: (a) when cerebella were taken from mice not older than one day of age: (b) when cells were seeded at higher plating densities; and (c) cultured in chemically defined medium which facilitates the survival of neurons. No Purkinje cells could be detected in cultures from mice older than 6 days. PC1 antigen expression developed in vitro on the same time scale as in vivo, i.e. it was first detectable at the equivalent of postnatal days 3–4. At this stage cell bodies had a size of 13–14 μm in diameter and few processes. Dendrite-like arborizations, with more than one primary dendrite, extension of usually only one thin and long (0.5–1.6 mm) axon-like process and collaterals directed preferentially towards other Purkinje cells, developed with time in culture until the final form was reached by the equivalent of approximately day 16. Cell body size was 18–19 μm in diameter at this stage. Cell shapes were reminiscent of those described in certain cerebellar mouse mutants and in experimentally produced agranular cerebella. Many ultrastructural features of these cells correlated with those described for the in vivo counterpart. However, there was a lack of spiny branchlets and abnormally long persisting somatic spines. Synaptic contacts of the ‘en passant’ type could be seen at the Purkinje cell soma. Gray type I synapses were seen on Purkinje cell dendrites and spines.