Primary Dissociated Cell Culture of Fetal Rat Central Nervous Tissue. I. Immunocytochemical and Ultrastructural Studies of Cell Development and Synaptogenesis

We have tried to establish a method of primary dissociated cell culture of the central nervous system (CNS) for successful development of large numbers of synapses and myelinated axons. Cerebra from 18-day-old fetal rats were enzymatically dissociated into single cells and plated onto poly-D-lysine-precoated coverslips at high cell density. With the progress of cell maturation, mixed neuronal and non-neuronal cell processes grew heavily and piled up on each other, making three-dimensional structures which corresponded to 'neuropil' in vivo. Within these structures we could observe not only many mature neurons and remarkable synaptogenesis but also many myelinated axons. The synapses were mainly axo-dendritic but axo-somatic synapses were also occasionally observed. Although most of the axon terminals contained many round clear vesicles which were about 30 nm in diameter, some of them contained both round clear vesicles and 50 nm in diameter vesicles with electron-dense cores. Also a small number of large electron-dense core vesicles (about 130 nm in diameter) were found in the perikarya of mature neurons. The numerous synapse formations observed in 3-dimensional structures suggest that neurons can remain in a stable state and carry out an active metabolism through neurotransmitters. So these structures are considered to provide a favorable microenvironment for both synaptogenesis and myelinogenesis.