Abstract
Insight into how glutamatergic synapses form in vivo is important for understanding developmental and experience-triggered changes of excitatory circuits. Here, we imaged postsynaptic densities (PSDs) expressing a functional, GFP-tagged glutamate receptor subunit (GluR-IIAGFP) at neuromuscular junctions of Drosophila melanogaster larvae for several days in vivo. New PSDs, associated with functional and structural presynaptic markers, formed independently of existing synapses and grew continuously until reaching a stable size within hours. Both in vivo photoactivation and photobleaching experiments showed that extrasynaptic receptors derived from diffuse, cell-wide pools preferentially entered growing PSDs. After entering PSDs, receptors were largely immobilized. In comparison, other postsynaptic proteins tested (PSD-95, NCAM and PAK homologs) exchanged faster and with no apparent preference for growing synapses. We show here that new glutamatergic synapses form de novo and not by partitioning processes from existing synapses, suggesting that the site-specific entry of particular glutamate receptor complexes directly controls the assembly of individual PSDs.
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References
Chklovskii, D.B., Mel, B.W. & Svoboda, K. Cortical rewiring and information storage. Nature 431, 782–788 (2004).
Gundelfinger, E.D., Kessels, M.M. & Qualmann, B. Temporal and spatial coordination of exocytosis and endocytosis. Nat. Rev. Mol. Cell Biol. 4, 127–139 (2003).
Washbourne, P., Bennett, J.E. & McAllister, A.K. Rapid recruitment of NMDA receptor transport packets to nascent synapses. Nat. Neurosci. 5, 751–759 (2002).
Bresler, T. et al. Postsynaptic density assembly is fundamentally different from presynaptic active zone assembly. J. Neurosci. 24, 1507–1520 (2004).
Niell, C.M. & Smith, S.J. Live optical imaging of nervous system development. Annu. Rev. Physiol. 66, 771–798 (2004).
Featherstone, D.E. & Broadie, K. Surprises from Drosophila: genetic mechanisms of synaptic development and plasticity. Brain Res. Bull. 53, 501–511 (2000).
Gramates, L.S. & Budnik, V. Assembly and maturation of the Drosophila larval neuromuscular junction. Int. Rev. Neurobiol. 43, 93–117 (1999).
Petersen, S.A., Fetter, R.D., Noordermeer, J.N., Goodman, C.S. & DiAntonio, A. Genetic analysis of glutamate receptors in Drosophila reveals a retrograde signal regulating presynaptic transmitter release. Neuron 19, 1237–1248 (1997).
DiAntonio, A., Petersen, S.A., Heckmann, M. & Goodman, C.S. Glutamate receptor expression regulates quantal size and quantal content at the Drosophila neuromuscular junction. J. Neurosci. 19, 3023–3032 (1999).
Marrus, S.B., Portman, S.L., Allen, M.J., Moffat, K.G. & DiAntonio, A. Differential localization of glutamate receptor subunits at the Drosophila neuromuscular junction. J. Neurosci. 24, 1406–1415 (2004).
Qin, G. et al. Four different subunits are essential for expressing the synaptic glutamate receptor at neuromuscular junctions of Drosophila. J. Neurosci. 25, 3209–3218 (2005).
Schuster, C.M., Davis, G.W., Fetter, R.D. & Goodman, C.S. Genetic dissection of structural and functional components of synaptic plasticity. I. Fasciclin II controls synaptic stabilization and growth. Neuron 17, 641–654 (1996).
Schuster, C.M. et al. Molecular cloning of an invertebrate glutamate receptor subunit expressed in Drosophila muscle. Science 254, 112–114 (1991).
Albin, S.D. & Davis, G.W. Coordinating structural and functional synapse development: postsynaptic p21-activated kinase independently specifies glutamate receptor abundance and postsynaptic morphology. J. Neurosci. 24, 6871–6879 (2004).
Sone, M. et al. Synaptic development is controlled in the periactive zones of Drosophila synapses. Development 127, 4157–4168 (2000).
Zito, K., Parnas, D., Fetter, R.D., Isacoff, E.Y. & Goodman, C.S. Watching a synapse grow: noninvasive confocal imaging of synaptic growth in Drosophila. Neuron 22, 719–729 (1999).
Broadie, K. & Bate, M. Activity-dependent development of the neuromuscular synapse during Drosophila embryogenesis. Neuron 11, 607–619 (1993).
Kawasaki, F., Zou, B., Xu, X. & Ordway, R.W. Active zone localization of presynaptic calcium channels encoded by the cacophony locus of Drosophila. J. Neurosci. 24, 282–285 (2004).
Campbell, R.E. et al. A monomeric red fluorescent protein. Proc. Natl. Acad. Sci. USA 99, 7877–7882 (2002).
Wucherpfennig, T., Wilsch-Brauninger, M. & Gonzalez-Gaitan, M. Role of Drosophila Rab5 during endosomal trafficking at the synapse and evoked neurotransmitter release. J. Cell Biol. 161, 609–624 (2003).
Kuromi, H. & Kidokoro, Y. Selective replenishment of two vesicle pools depends on the source of Ca2+ at the Drosophila synapse. Neuron 35, 333–343 (2002).
Heimbeck, G., Bugnon, V., Gendre, N., Haberlin, C. & Stocker, R.F. Smell and taste perception in Drosophila melanogaster larva: toxin expression studies in chemosensory neurons. J. Neurosci. 19, 6599–6609 (1999).
Marrus, S.B. & DiAntonio, A. Preferential localization of glutamate receptors opposite sites of high presynaptic release. Curr. Biol. 14, 924–931 (2004).
Takao-Rikitsu, E. et al. Physical and functional interaction of the active zone proteins, CAST, RIM1, and Bassoon, in neurotransmitter release. J. Cell Biol. 164, 301–311 (2004).
Ohtsuka, T. et al. Cast: a novel protein of the cytomatrix at the active zone of synapses that forms a ternary complex with RIM1 and munc13–1. J. Cell Biol. 158, 577–590 (2002).
Wang, Y., Liu, X., Biederer, T. & Sudhof, T.C. A family of RIM-binding proteins regulated by alternative splicing: Implications for the genesis of synaptic active zones. Proc. Natl. Acad. Sci. USA 99, 14464–14469 (2002).
Shapira, M. et al. Unitary assembly of presynaptic active zones from Piccolo-Bassoon transport vesicles. Neuron 38, 237–252 (2003).
Bachmann, A. et al. Cell type-specific recruitment of Drosophila Lin-7 to distinct MAGUK-based protein complexes defines novel roles for Sdt and Dlg-S97. J. Cell Sci. 117, 1899–1909 (2004).
Stewart, B.A., Schuster, C.M., Goodman, C.S. & Atwood, H.L. Homeostasis of synaptic transmission in Drosophila with genetically altered nerve terminal morphology. J. Neurosci. 16, 3877–3886 (1996).
Chen, K. & Featherstone, D.E. Discs-large (DLG) is clustered by presynaptic innervation and regulates postsynaptic glutamate receptor subunit composition in Drosophila. BMC Biol. 3, 1 (2005).
Schuster, C.M., Davis, G.W., Fetter, R.D. & Goodman, C.S. Genetic dissection of structural and functional components of synaptic plasticity. II. Fasciclin II controls presynaptic structural plasticity. Neuron 17, 655–667 (1996).
Patterson, G.H. & Lippincott-Schwartz, J. A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297, 1873–1877 (2002).
Sigrist, S.J. et al. Postsynaptic translation affects the efficacy and morphology of neuromuscular junctions. Nature 405, 1062–1065 (2000).
Ziv, N.E. & Garner, C.C. Principles of glutamatergic synapse formation: seeing the forest for the trees. Curr. Opin. Neurobiol. 11, 536–543 (2001).
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).
Fiala, J.C., Allwardt, B. & Harris, K.M. Dendritic spines do not split during hippocampal LTP or maturation. Nat. Neurosci. 5, 297–298 (2002).
Toni, N. et al. Remodeling of synaptic membranes after induction of long-term potentiation. J. Neurosci. 21, 6245–6251 (2001).
Sigrist, S.J., Thiel, P.R., Reiff, D.F. & Schuster, C.M. The postsynaptic glutamate receptor subunit DGluR-IIA mediates long-term plasticity in Drosophila. J. Neurosci. 22, 7362–7372 (2002).
Sigrist, S.J., Reiff, D.F., Thiel, P.R., Steinert, J.R. & Schuster, C.M. Experience-dependent strengthening of Drosophila neuromuscular junctions. J. Neurosci. 23, 6546–6556 (2003).
Shi, S., Hayashi, Y., Esteban, J.A. & Malinow, R. Subunit-specific rules governing AMPA receptor trafficking to synapses in hippocampal pyramidal neurons. Cell 105, 331–343 (2001).
Malinow, R. & Malenka, R.C. AMPA receptor trafficking and synaptic plasticity. Annu. Rev. Neurosci. 25, 103–126 (2002).
Borgdorff, A.J. & Choquet, D. Regulation of AMPA receptor lateral movements. Nature 417, 649–653 (2002).
Tardin, C., Cognet, L., Bats, C., Lounis, B. & Choquet, D. Direct imaging of lateral movements of AMPA receptors inside synapses. EMBO J. 22, 4656–4665 (2003).
Karunanithi, S., Marin, L., Wong, K. & Atwood, H.L. Quantal size and variation determined by vesicle size in normal and mutant Drosophila glutamatergic synapses. J. Neurosci. 22, 10267–10276 (2002).
Daniels, R.W. et al. Increased expression of the Drosophila vesicular glutamate transporter leads to excess glutamate release and a compensatory decrease in quantal content. J. Neurosci. 24, 10466–10474 (2004).
Budnik, V., Zhong, Y. & Wu, C.F. Morphological plasticity of motor axons in Drosophila mutants with altered excitability. J. Neurosci. 10, 3754–3768 (1990).
Renger, J.J., Ueda, A., Atwood, H.L., Govind, C.K. & Wu, C.F. Role of cAMP cascade in synaptic stability and plasticity: ultrastructural and physiological analyses of individual synaptic boutons in Drosophila memory mutants. J. Neurosci. 20, 3980–3992 (2000).
Ang, L.H., Kim, J., Stepensky, V. & Hing, H. Dock and Pak regulate olfactory axon pathfinding in Drosophila. Development 130, 1307–1316 (2003).
Aberle, H. et al. Wishful thinking encodes a BMP type II receptor that regulates synaptic growth in Drosophila. Neuron 33, 545–558 (2002).
Morin, X., Daneman, R., Zavortink, M. & Chia, W. A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila. Proc. Natl. Acad. Sci. USA 98, 15050–15055 (2001).
Acknowledgements
We thank H. Jäckle, G. Stuart and J. Eilers for comments on the manuscript; A. Schönle, J. Rietdorf, S. Höning and D. Sandstrom for advice; H. Aberle, U. Thomas, R. Ordway, E. Buchner, N. Harding, A. DiAntonio and R. Tsien for fly stocks and reagents; M. Richter for technical assistance and the screening team of Deutsche Forschungsgemeinschaft (DFG) SPP1111 Cell Polarity. This work was supported by grants from the DFG to S.J.S. and M.H.
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Rasse, T., Fouquet, W., Schmid, A. et al. Glutamate receptor dynamics organizing synapse formation in vivo. Nat Neurosci 8, 898–905 (2005). https://doi.org/10.1038/nn1484
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DOI: https://doi.org/10.1038/nn1484
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