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Visualizing synapse formation in arborizing optic axons in vivo: dynamics and modulation by BDNF

Abstract

Dynamic developmental changes in axon arbor morphology may directly reflect the formation, stabilization and elimination of synapses. We used dual-color imaging to study, in the live, developing animal, the relationship between axon arborization and synapse formation at the single cell level, and to examine the participation of brain-derived neurotrophic factor (BDNF) in synaptogenesis. Green fluorescent protein (GFP)-tagged synaptobrevin II served as a marker to visualize synaptic sites in individual fluorescently labeled Xenopus optic axons. Time-lapse confocal microscopy revealed that although most synapses remain stable, synapses are also formed and eliminated as axons branch and increase their complexity. Most new branches originated at GFP-labeled synaptic sites. Increasing BDNF levels significantly increased both axon arborization and synapse number, with BDNF increasing synapse number per axon terminal. The ability to visualize central synapses in real time provides insights about the dynamic mechanisms underlying synaptogenesis, and reveals BDNF as a modulator of synaptogenesis in vivo.

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Figure 1: GFP-synaptobrevin expression in live Xenopus laevis tadpoles.
Figure 2: GFP-synaptobrevin localizes to sites of synaptic membrane recycling and sites of contact between retinal and tectal neurons in culture.
Figure 3: GFP-synaptobrevin localizes to synaptic contact sites in vivo.
Figure 4: Dynamics of arborization and synaptogenesis in individual RGC axons.
Figure 5: GFP-synaptobrevin clusters are added and stabilized in arborizing RGC axons while new branches originate at GFP-labeled synaptic sites.
Figure 6: BDNF increases RGC axon arborization and GFP-synaptobrevin puncta in vivo.
Figure 7: BDNF specifically enhances synapse formation in addition to increasing axon arborization.

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Acknowledgements

We thank X.-h.Wang and M.-m. Poo for the GFP-Xsyb plasmid and B. Lom for help with initial experiments. We also thank A. Lontok for technical assistance and R. Frostig, B. Lom and D. Bok for discussions and comments on this manuscript. Supported by awards from the Arnold and Mabel Beckman Foundation and NIH (EY11912).

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Correspondence to Susana Cohen-Cory.

Supplementary information

Supplementary Figure 1

Montage of six consecutive confocal optical sections of a portion of an arbor double-labeled with GFP-synaptobrevin and DiI (shown in Fig. 4b, top middle panel). The montage illustrates the distribution of GFP-synaptobrevin in individual, thin optical sections. GFP-synaptobrevin clusters (yellow; red and green fluorescence overlay) are highly localized to individual optical sections. Arrowheads, arbor sites with high red fluorescent signal where a GFP-synaptobrevin cluster appears only in one optical section. Asterisks, thin portions of the arbor that exhibit GFP-synaptobrevin puncta. Black arrow, thick portion of the arbor where no GFP-synaptobrevin label is observed in any of the three consecutive optical sections. Black arrowhead, thick branch point that does not exhibit GFP-synaptobrevin labeling in any of the three optical sections. These individual optical sections clearly illustrate that GFP-synaptobrevin clusters are specific and not a result of the summation of the GFP label in thick portions of the arbor. (JPG 87 kb)

Supplementary Figure 2

Two-dimensional reconstructions of double-labeled RGC axon arbors imaged over time by confocal microscopy illustrate the distribution of the GFP-synaptobrevin and DsRed labels along the axon terminal. Here, by separating the green and red components, the intensity of the DsRed fluorescent signal appears more homogeneous than GFP-synaptobrevin label throughout the axon terminal (including branch points). (JPG 34 kb)

Supplementary Figure 3

Montage of individual confocal optical sections of axon arbors labeled with GFP-synaptobrevin and DiI and the projection of those sections into one plane demonstrate no significant cross talk between the fluorescent channels when imaging GFP and DiI or DsRed double-labeled axons. In this example, a GFP-synaptobrevin and DiI double-labeled axon is surrounded by a number of axons brightly labeled with DiI (red), or GFP-synaptobrevin (green), only. As evident from the individual confocal optical sections, GFP fluorescence is clearly distinguishable from DiI fluorescence. True coincidence in the GFP-synaptobrevin and DiI labels can only be observed in the double-labeled axon (yellow), both in thin as well as thick portions of the arbor (also illustrated in Fig. 4b). Non-specific yellow label can be generated however, when images are projected into one plane. Thus, we analyzed all our data by assessing individual optical sections. (JPG 81 kb)

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Alsina, B., Vu, T. & Cohen-Cory, S. Visualizing synapse formation in arborizing optic axons in vivo: dynamics and modulation by BDNF. Nat Neurosci 4, 1093–1101 (2001). https://doi.org/10.1038/nn735

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