Development of apparent presynaptic elements formed in response to polylysine coated surfaces

Formation of apparent presynaptic elements on polylysine-coated surfaces was examined with both scanning and transmission electron microscopy. Neurons from 2- or 8-day-old rat cerebellums were used in dissociated cell cultures as a source of growing neurites. An apparent presynaptic element is a defined swelling on a neurite with 40 nm diameter vesicles accumulated at the membrane which has a slight thickening. Apparent presynaptic elements do not have a normal postsynaptic element. In place of the postsynaptic element was the polylysine-coated surface of a large diameter Sepharose bead. The first apparent presynaptic elements were seen at 2 h of incubation, suggesting that morphologically identifiable synapses may form in this short a time. The number of apparent presynaptic elements on beads increased from 2 h to 24 h incubation and decreased from 5 to 9 days incubation. At all times non-neuronal cells grew up on to the beads and often covered both neurites and apparent presynaptic elements. In the longer incubations degenerating apparent presynaptic elements were seen engulfed by non-neuronal cells, suggesting that non-neuronal cells may have the ability to remove presynaptic elements that are not functioning. The number of synaptic vesicles per apparent presynaptic elements increased continuously between 2 h and 9 day incubation, eventually surpassing the number of synaptic vesicles seen in other presynaptic elements in cultures. This result suggests that an interaction between presynaptic and postsynaptic elements may be necessary to limit the number of synaptic vesicles found in presynaptic elements. Cultures grown for only 5 days in vitro (DIV) and incubated for 1 day with coated beads had the most apparent presynaptic elements, while those at 28 DIV and incubated for 1 day had the least. In the cultures at 5 DIV, neurons formed many apparent presynaptic elements, but these neurons could form only a very few normal synapses between themselves. Thus the beginning of the formation of synaptic contacts may depend on the availability of future postsynaptic elements and not on the lack of future presynaptic elements.