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In vivo imaging of synapse formation on a growing dendritic arbor

Abstract

The form of a neuron's dendritic arbor determines the set of axons with which it may form synaptic contacts, thus establishing connectivity within neural circuits. However, the dynamic relationship between dendrite growth and synaptogenesis is not well understood. To observe both processes simultaneously, we performed long-term imaging of non-spiny dendritic arbors expressing a fluorescent postsynaptic marker protein as they arborized within the optic tectum of live zebrafish larvae. Our results indicate that almost all synapses form initially on newly extended dendritic filopodia. A fraction of these nascent synapses are maintained, which in turn stabilizes the subset of filopodia on which they form. Stabilized filopodia mature into dendritic branches, and successive iterations of this process result in growth and branching of the arbor. These findings support a 'synaptotropic model' in which synapse formation can direct dendrite arborization.

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Figure 1: Imaging of individual cells on consecutive days reveals that punctum formation and dendrite growth are concurrent processes.
Figure 2: Long-term imaging of arbor growth and punctum formation.
Figure 3: Analysis of 'punctum-centric' movies reveals a typical mode of dendrite growth and puncta formation.
Figure 4: Dendrite growth occurs by an iterative sequence of selective filopodial stabilization and punctum formation.
Figure 5: PSD puncta and selective stabilization of dendritic filopodia.
Figure 6: Model of synaptotropic guidance of dendrite growth.

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Acknowledgements

We thank B. Barres, L. Luo, W. Talbot and R. Tsien for their comments on the manuscript. GAL4-VP16/UAS expression constructs were graciously provided by S. Fraser. This work was supported by the Wellcome Trust (M.P.M.), Howard Hughes Medical Institute (C.M.N.), the Vincent and Stella Coates Foundation and the National Institute of Neurological Disorders and Stroke (NS043461).

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Correspondence to Stephen J Smith.

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Supplementary information

Supplementary Fig. 1

Wholemount SV2 labeling of zebrafish tectal neuropil. Due to high synapse density, this technique could not reliably be used to demonstrate specific colocalization of PSD-95:GFP puncta with presynaptic SV2 puncta. Scale bar 5 μm. (JPG 59 kb)

Supplementary Fig. 2

Synaptophysin labelling (red channel) of dissociated hippocampal cells (6 d.i.v.) expressing zebrafish PSD-95:GFP (green channel) shows that 86% of PSD-95:GFP puncta colocalize with a synaptophysin punctum. (n = 456 PSD-95:GFP puncta from 19 cells). Scale bar 10μm. (JPG 39 kb)

Supplementary Fig. 3

Comparison of dendritic branch length over several days between cells expressing the PSD-95:GFP + DsRed construct or simply GFP reveals that neither PSD-95:GFP nor DsRed significantly affects dendrite growth (n = 6 cells each). (JPG 23 kb)

Supplementary Video 1

Timelapse of 3 day post-fertilization tectal cell dendrite expressing actin:GFP and soluble DsRed, shows a large number of highly motile, mostly transient, actin-based protrusions. Frames are at 3 minute intervals, total time 2 hours. (MOV 2371 kb)

Supplementary Video 2

Timelapse series which provided still images for Figure 2a in the text, showing net growth of the arbor and formation of stable PSD-95:GFP puncta. Frames are at 20 minute intervals, total time 20 hours. (MOV 2835 kb)

Supplementary Video 3

Timelapse series which provided still images for Figure 2b in the text, showing highly dynamic filopodia and transient PSD-95:GFP puncta. Frames are at 20 minute intervals, total time 12 hours. (MOV 1189 kb)

Supplementary Video 4

Timelapse series which provided still images for Figure 3 in the text, showing a typical mode of punctum formation on a filopodium. Frames are at 20 minute intervals, total time 6 hours. (MOV 823 kb)

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Niell, C., Meyer, M. & Smith, S. In vivo imaging of synapse formation on a growing dendritic arbor. Nat Neurosci 7, 254–260 (2004). https://doi.org/10.1038/nn1191

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