A transgenic zebrafish model for in vivo long-term imaging of retinotectal synaptogenesis.

The retinotectal synapse in larval zebrafish, combined with live time-lapse imaging, provides an advantageous model for study of the development and remodelling of central synapses in vivo. In previous studies, these synapses were labelled by transient expression of fluorescence-tagged synaptic proteins, which resulted in the dramatic variation of labelling patterns in each larva. Here, using GAL4-Upstream Activating Sequence (GAL4-UAS) methodology, we generated stable transgenic lines, which express EGFP-tagged synaptophysin (a presynaptic protein) in retinal ganglion cells (RGCs), to reliably label the pre-synaptic site of retinotectal synapses. This tool avoids the variable labelling of RGCs that occurs in transient transgenic larvae. We obtained several stable transgenic lines that differ consistently in the number of labelled RGCs. Using stable lines that consistently had a single labelled RGC, we could trace synaptogenic dynamics on an individual RGC axonal arbor across different developmental stages. In the stable lines that consistently had multiple labelled RGCs, we could simultaneously monitor both pre- and post-synaptic compartments by combining transient labelling of post-synaptic sites on individual tectal neurons. These tools allowed us to investigate molecular events underlying synaptogenesis and found that the microRNA-132 (miR-132) is required for developmental synaptogenesis. Thus, these transgenic zebrafish stable lines provide appropriate tools for studying central synaptogenesis and underlying molecular mechanisms in intact vertebrate brain.