The Journal of Neuroscience, March 29, 2006, ():
Evidence from In Vivo Imaging That Synaptogenesis Guides the Growth and Branching of Axonal Arbors by Two Distinct Mechanisms
J. Neurosci. Meyer and Smith
26: 3604
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplementary figure 1. In situ hybridization analysis shows that Synaptophysin mRNA is expressed broadly in the zebrafish nervous system at 3 dpf. (A) Lateral and (B) ventral view of wholemount fish labeled with antisense synaptophysin probe. (C, D and E) Transverse vibratome sections of wholemount labeled fish showing strong expression of Synaptophysin mRNA in the inner and outer plexiform layers of the retina (ipl and opl respectively), midbrain marginal zone (mz) and ventro-lateral spinal cord (sc). The expression pattern suggests that Synaptophysin mRNA is targeted to neuronal processes. (F and G) Wholemount fish processed with Synaptophysin sense control probe.
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Supplementary figure 2. Syp:GFP localizes to synaptic sites in vivo. (A). Confocal image of SV2 labeling of zebrafish tectal neuropil. Due to high synapse density in the tectum, this technique could not reliably be used to demonstrate specific colocalization of Syp:GFP puncta with presynaptic SV2 puncta. (B) In regions of the developing spinal cord where SV2 labeling was less dense we were able to demonstrate precise colocalization of SV2 and Syp:GFP puncta. In all figures a lateral view of the spinal cord is shown. Anterior is to the top, and dorsal is to the left. Co-localization of Syp:GFP (left panel) with SV2 immunolabeling (center panel) in zebrafish spinal neurons is demonstrated by overlaying the Syp:GFP and SV2 images (right panel). (C) To examine the degree of colocalization of Syp:GFP clusters with postsynaptic structures, Syp:GFP was expressed in spinal motor neurons, and labeled postsynaptic acetylcholine receptors were labeled with Alexa 594-tagged α-bungarotoxin. Two examples of spinal motor neurons expressing Syp:GFP (left panels), labeled acetylecholine receptors are shown in the centre panels.. Colocalization of Syp:GFP clusters with postsynaptic acetylcholine receptors are shown by overlaying the images from the left and center panels (right panel). Scale bar represents 5µm.
In spinal motor neurons of 2-day-old larvae, 86%±3.6% of Syp:GFP puncta colocalized with postsynaptic acetylcholine receptors (208 puncta from 7 cells) (Fig. 1B). At 2 days post-fertilization (dpf) motor neurons are still elaborating axonal arbors, thus even in developing axons the majority of Syp:GFP puncta colocalize with postsynaptic structures. The remaining AChR-free Syp:GFP puncta may be presynaptic structures prior to assembly of postsynaptic AChR clusters or presynaptic structures remaining from synapses in the process of disassembly. To verify the accuracy of TR-αBTX, and SV2 labeling with colocalization with Syp:GFP clusters, we compared colocalization measured in maximum intensity projections with optical sections in two-photon images for all Syp:GFP clusters We find that all of Syp:GFP puncta scored as synapses in maximum intensity projections also colocalize with TR-αBTX and SV2 labeling within the same optical section in z series. This indicates that our analysis in flattened images accurately demonstrates colocalization of Syp:GFP accumulations with both presynaptic SV2 puncta and postsynaptic AChR clusters. Lastly, in RGCs there was no systematic variation in the density or intensity of Syp:GFP puncta with distance from the soma, indicating that the GFP puncta we observed are not nonspecific aggregations resulting from overexpression.
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Supplementary video 1. Zebrafish RGC expressing Syp:GFP-DSR as it arborizes in the optic tectum. Imaging was started at 3 dpf. Images were taken at 10 min intervals for a period of 10 hours.
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Supplementary video 2. Movie of 3 dpf RGC axon expressing Syp:GFP-DSR, showing puncta forming in the wake of growth cones and on axonal filopodia as the cell arborizes in the optic tectum. Note also growth cone splitting events. Images were taken at 10 min intervals for 10 h.
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Supplementary video 3. Cropped view of 3dpf RGC axon showing puncta
forming on filopodia. Imaging interval is 10 min. Movie represents 8.5 h of real
time.
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Supplementary video 4. Cropped view of 3dpf RGC axon showing that
branch elimination occurs gradually in a synapse-to-synapse, stepwise manner. Note dimming of Syp:GFP cluster prior to the onset of retraction, suggesting that synapse disassembly precedes branch retraction. Imaging interval is 10 min. Movie represents 10 h of real time.
