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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Ramoa, A. S., Campbell, G. & Shatz, C. J. Dendritic growth and remodeling of cat retinal ganglion cells during fetal and postnatal development. J. Neurosci. 8, 4239–4261 (1988).
Rourke, N. A. & Fraser, S. E. Dynamic changes in optic fiber terminal arbors lead to retinotopic map formation: an in vivo confocal microscopic study. Neuron 5, 159–171 (1990).
Antonini, A. & Stryker, M. P. Rapid remodeling of axonal arbors in the visual cortex. Science 260, 1819–1821 (1993).
Katz, L. C. & Shatz, C. J. Synaptic activity and the construction of cortical circuits. Science 274, 1133–1138 (1996).
Constantine-Paton, M. NMDA receptor as a mediator of activity-dependent synaptogenesis in the developing brain. Cold Spring Harb. Symp. Quant. Biol. 55, 431–443 (1990).
Lendvai, B., Stern, E. A., Chen, B. & Svoboda, K. Experience-dependent plasticity of dendritic spines in the developing rat barrel cortex in vivo. Nature 404, 876–881 (2000).
Snider, W. D. & Lichtman, J. W. Are neurotrophins synaptotrophins? Mol. Cell Neurosci. 7, 433–442 (1996).
McAllister, A. K., Katz, L. C. & Lo, D. C. Neurotrophins and synaptic plasticity. Annu. Rev. Neurosci. 22, 295–318 (1999).
Schuman, E. M. Neurotrophin regulation of synaptic transmission. Curr. Opin. Neurobiol. 9, 105–109 (1999).
Cohen-Cory, S. & Fraser, S. E. Effects of brain-derived neurotrophic factor on optic axon branching and remodelling in vivo. Nature 378, 192–196 (1995).
McAllister, A. K., Lo, D. C. & Katz, L. C. Neurotrophins regulate dendritic growth in developing visual cortex. Neuron 15, 791–803 (1995).
Inoue, A. & Sanes, J. R. Lamina-specific connectivity in the brain: regulation by N-cadherin, neurotrophins, and glycoconjugates. Science 276, 1428–1431 (1997).
Lom, B. & Cohen-Cory, S. Brain-derived neurotrophic factor differentially regulates retinal ganglion cell dendritic and axonal arborization in vivo. J. Neurosci. 19, 9928–9938 (1999).
Lohof, A. M., Ip, N. Y. & Poo, M. M. Potentiation of developing neuromuscular synapses by the neurotrophins NT-3 and BDNF. Nature 363, 350–353 (1993).
Kang, H. & Schuman, E. M. Long-lasting neurotrophin-induced enhancement of synaptic transmission in the adult hippocampus. Science 267, 1658–1662 (1995).
Kafitz, K. W., Rose, C. R., Thoenen, H. & Konnerth, A. Neurotrophin-evoked rapid excitation through TrkB receptors. Nature 401, 918–921 (1999).
Rutherford, L. C., Nelson, S. B. & Turrigiano, G. G. BDNF has opposite effects on the quantal amplitude of pyramidal neuron and interneuron excitatory synapses. Neuron 21, 521–530 (1998).
Vicario-Abejon, C., Collin, C., McKay, R. D. & Segal, M. Neurotrophins induce formation of functional excitatory and inhibitory synapses between cultured hippocampal neurons. J. Neurosci. 18, 7256–7271 (1998).
Seil, F. J. & Drake-Baumann, R. TrkB receptor ligands promote activity-dependent inhibitory synaptogenesis. J. Neurosci. 20, 5367–5373 (2000).
Cabelli, R. J., Hohn, A. & Shatz, C. J. Inhibition of ocular dominance column formation by infusion of NT-4/5 or BDNF. Science 267, 1662–1666 (1995).
Huang, Z. J. et al. BDNF regulates the maturation of inhibition and the critical period of plasticity in mouse visual cortex. Cell 98, 739–755 (1999).
Causing, C. G. et al. Synaptic innervation density is regulated by neuron-derived BDNF. Neuron 18, 257–267 (1997).
Martinez, A. et al. TrkB and TrkC signaling are required for maturation and synaptogenesis of hippocampal connections. J. Neurosci. 18, 7336–7350 (1998).
Gonzalez, M. et al. Disruption of TrkB-mediated signaling induces disassembly of postsynaptic receptor clusters at neuromuscular junctions. Neuron 24, 567–583 (1999).
Pozzo-Miller, L. D. et al. Impairments in high-frequency transmission, synaptic vesicle docking, and synaptic protein distribution in the hippocampus of BDNF knockout mice. J. Neurosci. 19, 4972–4983 (1999).
Zou, D. J. & Cline, H. T. Postsynaptic calcium/calmodulin-dependent protein kinase II is required to limit elaboration of presynaptic and postsynaptic neuronal arbors. J. Neurosci. 19, 8909–8918 (1999).
Dingwell, K. S., Holt, C. E. & Harris, W. A. The multiple decisions made by growth cones of RGCs as they navigate from the retina to the tectum in Xenopus embryos. J. Neurobiol. 44, 246–259 (2000).
Cohen-Cory, S. BDNF modulates, but does not mediate, activity-dependent branching and remodeling of optic axon arbors in vivo. J. Neurosci. 19, 9996–10003 (1999).
Trimble, W. S., Cowan, D. M. & Scheller, R. H. VAMP-1: a synaptic vesicle-associated integral membrane protein. Proc. Natl. Acad. Sci. USA 85, 4538–4542 (1988).
Ahmari, S. E., Buchanan, J. & Smith, S. J. Assembly of presynaptic active zones from cytoplasmic transport packets. Nat. Neurosci. 3, 445–451 (2000).
Nonet, M. L. Visualization of synaptic specializations in live C. elegans with synaptic vesicle protein-GFP fusions. J. Neurosci. Methods 89, 33–40 (1999).
Zhen, M. & Jin, Y. The liprin protein SYD-2 regulates the differentiation of presynaptic termini in C. elegans. Nature 401, 371–375 (1999).
Sollner, T. & Rothman, J. E. Neurotransmission: harnessing fusion machinery at the synapse. Trends Neurosci. 17, 344–348 (1994).
Matz, M. V. et al. Fluorescent proteins from nonbioluminescent Anthozoa species. Nat. Biotechnol. 17, 969–973 (1999).
Holt, C. E., Garlick, N. & Cornel, E. Lipofection of cDNAs in the embryonic vertebrate central nervous system. Neuron 4, 203–214 (1990).
Betz, W. J. & Bewick, G. S. Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction. Science 255, 200–203 (1992).
Ziv, N. E. & Smith, S. J. Evidence for a role of dendritic filopodia in synaptogenesis and spine formation. Neuron 17, 91–102 (1996).
Kistner, U. et al. SAP90, a rat presynaptic protein related to the product of the Drosophila tumor suppressor gene dlg-A. J. Biol. Chem. 268, 4580–4583 (1993).
Fletcher, T. L., Cameron, P., De Camilli, P. & Banker, G. The distribution of synapsin I and synaptophysin in hippocampal neurons developing in culture. J. Neurosci. 11, 1617–1626 (1991).
Jontes, J. D., Buchanan, J. & Smith, S. J. Growth cone and dendrite dynamics in zebrafish embryos: early events in synaptogenesis imaged in vivo. Nat. Neurosci. 3, 231–237 (2000).
Friedman, H. V., Bresler, T., Garner, C. C. & Ziv, N. E. Assembly of new individual excitatory synapses: time course and temporal order of synaptic molecule recruitment. Neuron 27, 57–69 (2000).
Alder, J., Kanki, H., Valtorta, F., Greengard, P. & Poo, M. M. Overexpression of synaptophysin enhances neurotransmitter secretion at Xenopus neuromuscular synapses. J. Neurosci. 15, 511–519 (1995).
Toni, N., Buchs, P. A., Nikonenko, I., Bron, C. R. & Muller, D. LTP promotes formation of multiple spine synapses between a single axon terminal and a dendrite. Nature 402, 421–425 (1999).
Campbell, G. & Shatz, C. J. Synapses formed by identified retinogeniculate axons during the segregation of eye input. J. Neurosci. 12, 1847–1858 (1992).
Yen, L., Sibley, J. T. & Constantine-Paton, M. Analysis of synaptic distribution within single retinal axonal arbors after chronic NMDA treatment. J. Neurosci. 15, 4712–4725 (1995).
Pinches, E. M. & Cline, H. T. Distribution of synaptic vesicle proteins within single retinotectal axons of Xenopus tadpoles. J. Neurobiol. 35, 426–434 (1998).
Silver, M. A. & Stryker, M. P. Synaptic density in geniculocortical afferents remains constant after monocular deprivation in the cat. J. Neurosci. 19, 10829–10842 (1999).
Norden, J. J. & Constantine-Paton, M. Dynamics of retinotectal synaptogenesis in normal and 3-eyed frogs: evidence for the postsynaptic regulation of synapse number. J. Comp. Neurol. 348, 461–479 (1994).
Balice-Gordon, R. J., Chua, C. K., Nelson, C. C. & Lichtman, J. W. Gradual loss of synaptic cartels precedes axon withdrawal at developing neuromuscular junctions. Neuron 11, 801–815 (1993).
Nieuwkoop, P. D. & Faber, J. Normal Table of Xenopus laevis (Elsevier North Holland, Amsterdam, The Netherlands, 1956).
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).
Author information
Authors and Affiliations
Corresponding author
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)
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nn735
This article is cited by
-
DSCAM is differentially patterned along the optic axon pathway in the developing Xenopus visual system and guides axon termination at the target
Neural Development (2022)
-
Distinct Effects of BDNF and NT-3 on the Dendrites and Presynaptic Boutons of Developing Olfactory Bulb GABAergic Interneurons In Vitro
Cellular and Molecular Neurobiology (2022)
-
Can the administration of platelet lysates to the brain help treat neurological disorders?
Cellular and Molecular Life Sciences (2022)
-
Effects of 7,8-Dihydroxyflavone on Lipid Isoprenoid and Rho Protein Levels in Brains of Aged C57BL/6 Mice
NeuroMolecular Medicine (2021)
-
Neurotrophic effects of GM1 ganglioside, NGF, and FGF2 on canine dorsal root ganglia neurons in vitro
Scientific Reports (2020)