Assembly of presynaptic active zones from cytoplasmic transport packets

Little is known about presynaptic assembly during central nervous system sy naptogenesis. Here we used time-lapse fluorescence imaging, immunocytochemi stry and electron microscopy to study hippocampal neuronal cultures transfe cted with a fusion construct of the presynaptic vesicle protein VAMP and gr een fluorescent protein. Our results suggest that major cytoplasmic and mem brane-associated protein precursors of the presynaptic active zone are tran sported along developing axons together as discrete packets. Retrospective electron microscopy demonstrated varied vesicular and tubulovesicular membr ane structures. Packets containing these heterogeneous structures were stab ilized specifically at new sites of dendrite- and axon-initiated cell-cell contact; within less than one hour, evoked vesicle recycling was observed a t these putative nascent synapses. These observations suggest that substant ial membrane remodeling may be necessary to produce the uniform vesicles ty pical of the mature active zone, and that many presynaptic proteins may be united early in their biogenesis and sorting pathways.