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Mixed-culture assays for analyzing neuronal synapse formation

Nature Protocols volume 2, pages 670–676 (2007)Cite this article

Abstract

The assembly of synapses in the vertebrate central nervous system requires bidirectional signaling across the synaptic cleft that directs the differentiation of pre- and postsynaptic membrane domains. Biochemical and genetic studies have identified several adhesion and signaling molecules that localize to synapses and might participate in organizing synaptic structures. Understanding how individual proteins contribute to synaptic organization is complicated by the fact that there are significant numbers of separate signals that cooperate in this process. This protocol describes an assay system that permits examination of synaptogenic activities of individual cell-surface proteins in isolation. Besides the time needed for preparation and growth of primary neuronal cultures (6–14 days), the execution and analysis of the assay is rapid, requiring approximately 2 days. Using this assay, recent studies revealed that single synaptic adhesion complexes can direct a remarkable degree of synaptic differentiation and provided new insights into the cell biological mechanisms of synaptogenesis.

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Figure 1: Co-culture design.

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Acknowledgements

T.B. acknowledges the advice and support he received while collaborating with Dr. Ege Kavalali, UT Southwestern Medical Center, Dallas, on FM imaging and electrophysiological analyses of the mixed-culture system and thanks Massimiliano Stagi for manuscript comments. P.S. thanks members of his laboratory for comments on the manuscript. Work in the laboratory of P.S. was supported by R01 NS532830 and that in the laboratory of T.B. by R01 DA018928.

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Authors and Affiliations

  1. Department of Molecular Biophysics and Biochemistry, Yale University, Sterling Hall of Medicine C127, 333 Cedar Street, New Haven, 06520, Connecticut, USA

    Thomas Biederer

  2. Department of Physiology and Cellular Biophysics, Center for Neurobiology and Behavior, Columbia University, 630 West 168th Street, P&S 11-511, MC 22, New York, 10032, New York, USA

    Peter Scheiffele

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  1. Thomas Biederer
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  2. Peter Scheiffele
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Correspondence to Thomas Biederer or Peter Scheiffele.

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Biederer, T., Scheiffele, P. Mixed-culture assays for analyzing neuronal synapse formation. Nat Protoc 2, 670–676 (2007). https://doi.org/10.1038/nprot.2007.92

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