Fig. 5. Distribution and dynamics of GFP-synaptobrevin labeled presynaptic sites along RGC axon terminals. (A) Time-lapse sequence of a region of a control arbor illustrates the dynamic relationship between presynaptic site location and axon branch formation. New axonal branches originate from sites rich in GFP-synaptobrevin puncta (arrowheads), while new GFP-synaptobrevin clusters appear along an axon branch (Alsina et al., 2001). (B) Magnified region of an arbor illustrates the localization of GFP-synaptobrevin puncta to a nascent branch (arrows) in a DsRed2 labeled axon and its disappearance after anti-BDNF treatment (overlay, top panel; GFP-synaptobrevin fluorescence only, bottom panel). In some branches, GFP-synaptobrevin cluster dismantling precedes axon branch elimination (arrow), as indicated by the significant decrease in GFP fluorescence at the 2 hour time point. (C) Time-lapse sequence of a region of an anti-BDNF treated axon arbor shows the disappearance of GFP-synaptobrevin clusters and the retraction of an axon branch (arrow). The arrowhead indicates a site where a decrease in punctuate GFP-synaptobrevin fluorescence correlates with the shortening of the distal region of the axon branch. Asterisks indicate arbor sites with stable GFP-synaptobrevin clusters. Scale bars: 20 µm in A; 10 µm in B,C. Posterior is upwards, anterior is downwards.