Synapse formation in hippocampal neurons

Fast neurotransmitter secretion occurs at active zones with a conspicuous specialization of the presynaptic plasma membrane and a cytoplasmic cluster of synaptic vesicles. Despite increasing knowledge of the steps involved in assembly of the neuromuscular junction, little is known about how functional, positionally stable active zones are assembled and disassembled during central nervous system (CNS) synaptogenesis. Using techniques including time-lapse fluorescence imaging, immunocytochemistry, and electron microscopy, I have examined this process.; Using young hippocampal neuronal cultures transfected with a fusion construct of the presynaptic vesicle protein VAMP and green fluorescent protein, I found that major cytoplasmic and membrane-associated protein presynaptic active zone precursors are transported along developing axons together as discrete aggregates I termed "transport packets". Retrospective electron microscopy of transport packets demonstrated varied vesicular and tubulovesicular membrane structures. Packets containing these heterogeneous structures were stabilized specifically at new sites of dendrite- and axon-initiated cell-cell contact; within less than one hour, evoked vesicle recycling was observed at these putative nascent synapses. These observations suggest that substantial membrane remodeling may be necessary to produce uniform vesicles typical of mature active zones, and that many presynaptic proteins may be united early in their biogenesis and sorting pathways.; Having found that transport packets are important in the generation of new presynaptic active zones, I examined the processes involved in the transformation of a transport packet into a mature presynaptic active zone. Maturation of hippocampal cultures led to a decline in the number of observed transport packets; this is consistent with stabilization of packets at new synaptic sites. In addition, stable VAMP-GFP puncta were more likely than transport packets to demonstrate evoked vesicle recycling using FM 4-64.; I next examined processes involved in synapse disassembly. I applied HAV peptides, which are thought to inhibit cadherin-mediated adhesion, to mature hippocampal cultures transfected with VAMP-GFP. This led to dispersal throughout the axon of the signal associated with stable VAMP-GFP puncta. Dispersal of VAMP-GFP was also seen when loss of axo-dendritic adhesion was directly observed using timelapse confocal microscopy. These data give evidence for a role for adhesion molecules in synapse formation and stabilization.