Evidence from in vivo imaging that synaptogenesis guides the growth and branching of axonal arbors by two distinct mechanisms

To explore the relationship between axon arbor growth and synaptogenesis, developing retinal ganglion cell (RGC) axon arbors in zebrafish optic tectum were imaged in vivo at high temporal and spatial resolution using two-photon microscopy. Individual RGC axons were dually labeled by expression of a cytosolic red fluorescent protein (DsRed Express) to mark arbor structure and a fusion of the synaptic vesicle protein synaptophysin with green fluorescent protein (Syp: GFP) to mark presynaptic vesicles. Analysis of time-lapse sequences acquired at 10 min intervals revealed unexpectedly rapid kinetics of both axon branch and vesicle cluster turnover. Nascent axonal branches exhibited short average lifetimes of 19 min, and only 17% of newly extended axonal processes persisted for periods exceeding 3 h. The majority (70%) of Syp: GFP puncta formed on newly extended axonal processes. Syp: GFP puncta also exhibited short average lifetimes of 30 min, and only 34% of puncta were stabilized for periods exceeding 3 h. Moreover, strongly correlated dynamics of Syp: GFP puncta and branch structure suggest that synaptogenesis exerts strong influences on both the extension and the selective stabilization of nascent branches. First, new branches form almost exclusively at newly formed Syp: GFP puncta. Second, stabilized nascent branches invariably bear Syp: GFP puncta, and the detailed dynamics of branch retraction suggest strongly that nascent synapses can act at branch tips to arrest retraction. These observations thus provide evidence that synaptogenesis guides axon arbor growth by first promoting initial branch extension and second by selective branch stabilization.