Morphological changes in the neuritic growth cone and target neuron during synaptic junction development in culture

Abstract

Our object was to characterize the morphological changes occurring in pre- and postsynaptic elements during their initial contact and subsequent maturation into typical synaptic profiles. Neurons from superior cervical ganglia (SCG) of perinatal rats were freed of their supporting cells and established as isolated cells in culture. To these were added explants of embryonic rat thoracic spinal cord to allow interaction between outgrowing cord neurites and the isolated autonomic neurons. Time of initial contact was assessed by light microscopy; at timed intervals thereafter, cultures were fixed for electron microscopy. Upon contact, growth cone filopodia became extensively applied to the SCG neuronal plasmalemma and manifested numerous punctate regions in which the apposing plasma membranes were separated by only 7-10 nm. The Golgi apparatus of the target neuron hypertrophied, and its production of coated vesicles increased. Similar vesicles were seen in continuity with the SCG plasmalemma near the close contact site; their apparent contribution of a region of postsynaptic membrane with undercoating was considered to be the first definitive sign of synapse formation. Tracer work with peroxidase and ferritin confirmed that the traffic of coated vesicles within the neuronal soma is largely from Golgi region to somal surface. Subsequent to the appearance of postsynaptic density, the form and content of the growth cone was altered by the loss of filopodia and the appearance of synaptic vesicles which gradually became clustered opposite the postsynaptic density. As the synapse matured, synaptic vesicles increased in number, cleft width and content increased, presynaptic density appeared, branched membranous reticulum became greatly diminished, and most lysosomal structures disappeared. Coated vesicles continued to be associated with the postsynaptic membrane at all stages of maturation. The incorporation of Golgi-derived vesicles into discrete regions of the cell membrane could provide the mechanism for confining specific characteristics of the neuronal membrane to the synaptic region.