Fig. 3

Use of FingRs in visualizing and tracking synapses.

(A) Lateral view of eye from transgenic zebrafish expressing FingR(PSD95)-GFP and RFP-caax in retinal ganglion neurons (transgenic Tg(isl2b:Gal4); Tg(UAS:RFP-caax); Tg(FingR(PSD95)-GFP)). GFP expression is seen in puncta (arrowheads) and neuron somas (arrow); RFP labels neurons, dendrites, and their axons as they project towards optic chiasm (Supplemental Movie 1). Confocal z-stacks at 72?hpf, immunohistochemistry for GFP and RFP, scale bar 10??m. (B) Dorsal view of tectum in Tg(isl2b:Gal4); Tg(UAS:RFP-caax); Tg(FingR(PSD95)-GFP) embryo. Arrow shows FingR(PSD95)-GFP puncta along RGC axon, but not along entire length of axon (arrowhead). Confocal z-stack at 72?hpf, immunohistochemistry for GFP and RFP; scale bar 50??m, 5??m in inset panel. (C) Larvae co-expressing FingR(PDS95)-GFP (open arrowheads) and FingR(GPHN)-Cherry (arrows) demonstrates presence of neighboring excitatory and inhibitory puncta. Confocal z-stacks; scale bar 10??m, 2.5??m in inset panel. (D,E) FingRs can be used for in vivo visualization and monitoring of synapses. (D) Time-series of dopaminergic neurons and synapses in Tg(otpb.A:Gal4); Tg(FingR(PSD95)-GFP) animal, demonstrating changes in synapse number and expression with development. Scale bar 20??m. (E) Time-series of dopaminergic neurons and synapses development in Tg(otpb.A:Gal4); Tg(FingR(GPHN)-Cherry) animal, demonstrating changes in synapse number and expression with development. Scale bar 20??m. (F) Dorsal view in trunk at 96?hpf showing that FingR(PSD95)-GFP is expressed in the spinal cord (arrowhead), but not in axon projections labeled with SV2 (open arrowhead) in the neuromuscular synapses when driven in motor neurons (arrow) by Hb9:Gal4. Confocal z-stacks at 96?hpf, dorsal views, immunohistochemistry for GFP and SV2; scale bar 50??m, 10??m in inset panel.