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Glutamate receptor dynamics organizing synapse formation in vivo

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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Figure 1: GluR-IIAGFP labels the PSDs of individual NMJ synapses.
Figure 2: GluR-IIAGFP is fully functional.
Figure 3: In vivo imaging of PSD formation and growth during NMJ development.
Figure 4: PSDs labeled by GluR-IIAGFP are associated with markers of presynaptic structure and function.
Figure 5: In vivo FRAP analysis for several proteins expressed at the postsynaptic site of the NMJ.
Figure 6: Visualization of glutamate receptor mobility during PSD formation in vivo.
Figure 7: Bleach area–dependence of glutamate receptor FRAP during synapse formation and growth.

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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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Correspondence to Manfred Heckmann or Stephan J Sigrist.

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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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