Fig. 1. GFP-synaptobrevin specifically localizes to presynaptic sites in RGC axon terminals. (A-D) The localization of GFP-synaptobrevin was determined by examining the distribution of GFP immunoreactivity by electron microscopy. Morphologically mature synapses (black arrows), containing presynaptic terminals with numerous synaptic vesicles (v) and clearly defined pre- and postsynaptic specializations, are present in the tectal neuropil of stage 45 tadpoles. (B-D) Electron photomicrographs of tadpole brains immunostained with an antibody to GFP show the localization of gold particles (open arrows) to presynaptic terminals in the tectal neuropil. Silver enhancement of the secondary antibody coupled to 1 nm gold particles shows that the GFP immunolabel is preferentially associated to synaptic vesicles in morphologically mature retinotectal synapses (B,D), as well as in presynaptic terminals near contact sites (C). Scale bar: 0.2 µm. (E) Regions of an individual RGC axon arbor imaged at 5 minute intervals illustrate the distribution of GFP-synaptobrevin puncta. The majority of the GFP-synaptobrevin puncta remain constant throughout time. This is in contrast to motile GFP-synaptobrevin puncta present in small transport packets, prevalent in axon terminals of neurons grown in culture (Ahmari et al., 2000). Scale bar: 20 µm.