EARLY SYNAPTOGENESIS IN-VITRO - ROLE OF AXON TARGET DISTANCE

In contrast to some previous reports suggesting a delay in synapse for mation in vitro, we found that under ideal conditions, most hippocampa l and hypothalamic rat neurons were synaptically coupled after 3 or 4 days in vitro. Synaptophysin immunocytochemistry revealed strongly sta ined presynaptic boutons by 3 days in vitro. Studies with time-lapse l aser confocal imaging of FM1-43 revealed that axonal boutons were recy cling their synaptic vesicles, an indication of synapse formation, as early as 3 days after plating. To test the hypothesis that neurite out growth was enhanced in high-density cultures, thereby increasing the p robability of synapse formation, neurons were transfected with the jel lyfish green fluorescent protein (GFP) gene. After 2 days in high-dens ity cultures, green fluorescent neurites were about three times longer than in sister neurons plated in low-density cultures. Even in single dishes, GFP-transfected cells in contact with other neurons had neuri tes that were at least three times longer and grew faster than more is olated cells. Neurons grew longer neurites (+51%) when growing on surf ace membranes of heat-killed neurons than on polylysine, underlining t he importance of plasma membrane contact. Calcium imaging with fura-2 and whole cell recording showed that both GABA and glutamate presynapt ic release occurred after 3 or 4 days in vitro in high-density culture s but was absent in low-density cultures at this time. Together, these morphological, cytochemical, and physiological data suggest that the distance an axon must grow to find a postsynaptic partner plays a subs tantial role in the timing of synapse formation. Although other factor s in vitro may also play a role, the distance to a postsynaptic target , which defines the interval during which an axon grows to its target, can probably account for much of the difference in timing of synapse formation previously reported in vitro. A short intercell distance may increase the concentration of limited amounts of trophic factors avai lable to a nearby cell, and once contact is made, a neuronal membrane provides a superior substrate for neuritic elongation. (C) 1998 Wiley- Liss, Inc.