Development and fine structure of murine Purkinje cells in dissociated cerebellar cultures: dendritic differentiation, synaptic maturation, and formation of cell-class specific features

Abstract

The morphological differentiation of E16 murine Purkinje cells (PCs) in dissociated cerebellar cultures was analyzed by light and electron microscopic immunocytochemistry after 2-5 weeks in vitro (wiv), with particular emphasis on dendritic differentiation, synaptic maturation, and formation of stereotypical fine structural features. This study complements a companion paper on the features of PCs after 1 wiv. After 2 wiv, the PCs have an eccentric nucleus and the cytoplasmic organelles appear immature; the axon has a distinct initial segment and beaded axon collaterals but its boutons still contain sparse synaptic vesicles; dendrites show few bifurcations and tufts of spiny branchlets. After 3 wiv, the PCs display a centered nucleus, an extensive hypolemmal cisternal system, and stacks of up to four cisterns of granular endoplasmic reticulum; there is an increased number of dendritic bifurcations, spiny branchlets, mature spines, and axonal branches; dendritic tips still contain vesicle clusters, suggesting growth, and many synapses and afferent boutons continue to display immature features. After 4 wiv, elaborate perinucleolar coiled body rosettes, subsurface cistern-mitochondrion complexes and large stacks of granular endoplasmic reticulum finally appear within the soma; dendrites show a further increase in the numbers of bifurcations, segments and spines; most spines are synaptic and show mature features; afferent synapses are differentially distributed; PC boutons consistently display mature features and show a considerable degree of target specificity, although naked spines and reduced glial sheaths persist. After 5 wiv, PCs do not show further maturation and some dystrophic features appear. We conclude that under standard conditions and despite the disruption of normal tissue organization, PCs in dissociated cultures differentiate maximally after 4 wiv, at which stage they display many of the light and electron microscopic features that characterize mature PCs in situ. This prolonged developmental time-frame resembles that in the normal cerebellum. In view of the increasing usage of dissociated cerebellar cultures to study aspects of neuronal differentiation, synaptic activation and neuronal-glial interactions, an elucidation of the neurocytology of dissociated cerebellar cultures as presented in this study provides important clues for the interpretation of experimental data.