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Culture of Mouse Giant Central Nervous System Synapses and Application for Imaging and Electrophysiological Analyses

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Neurotrophic Factors

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1727))

Abstract

Primary neuronal cell culture preparations are widely used to investigate synaptic functions. This chapter describes a detailed protocol for the preparation of a neuronal cell culture in which giant calyx-type synaptic terminals are formed. This chapter also presents detailed protocols for utilizing the main technical advantages provided by such a preparation, namely, labeling and imaging of synaptic organelles and electrophysiological recordings directly from presynaptic terminals.

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References

  1. Forsythe ID (1994) Direct patch recording from identified presynaptic terminals mediating glutamatergic EPSCs in the rat CNS, in vitro. J Physiol 479:381–387

    Article  PubMed  PubMed Central  Google Scholar 

  2. Borst JGG, Helmchen F, Sakmann B (1995) Pre- and postsynaptic whole-cell recordings in the medial nucleus of the trapezoid body of the rat. J Physiol 489:825–840

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Takahashi T, Forsythe ID, Tsujimoto T, Barnes-Davies M, Onodera K (1996) Presynaptic calcium current modulation by a metabotropic glutamate receptor. Science 274:594–597

    Article  CAS  PubMed  Google Scholar 

  4. Schneggenburger R, Forsythe ID (2006) The calyx of held. Cell Tissue Res 326:311–337

    Article  PubMed  Google Scholar 

  5. Matteoli M, Takei K, Perin MS, Südhof TC, De Camilli P (1992) Exo-endocytotic recycling of synaptic vesicles in develo** processes of cultured hippocampal neurons. J Cell Biol 117:849–861

    Article  CAS  PubMed  Google Scholar 

  6. O’Brien RJ, Xu D, Petralia RS, Steward O, Huganir RL, Worley P (1999) Synaptic clustering of AMPA receptors by the extracellular immediate-early gene product Narp. Neuron 23:309–323

    Article  PubMed  Google Scholar 

  7. Schoch S, Deák F, Königstorfer A, Mozhayeva M, Sara Y, Südhof TC, Kavalali ET (2001) SNARE function analyzed in synaptobrevin/VAMP knockout mice. Science 294:1117–1122.

    Google Scholar 

  8. El-Husseini AE-D e-D, Schnell E, Dakoji S, Sweeney N, Zhou Q, Prange O, Gauthier-Campbell C, Aguilera-Moreno A, Nicoll RA, Bredt DS (2002) Synaptic strength regulated by palmitate cycling on PSD-95. Cell 108:849–863

    Article  CAS  Google Scholar 

  9. Mauch DH, Nägler K, Schumacher S, Göritz C, Müller EC, Otto A, Pfrieger FW (2001) CNS synaptogenesis promoted by glia-derived cholesterol. Science 294:1354–1357

    Article  CAS  PubMed  Google Scholar 

  10. Dean C, Scholl FG, Choih J, DeMaria S, Berger J, Isacoff E, Scheiffele P (2003) Neurexin mediates the assembly of presynaptic terminals. Nat Neurosci 6:708–716

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Dimitrov D, Takagi H, Guillaud L, Saitoh N, Eguchi K, Takahashi T (2016) Reconstitution of Giant Mammalian Synapses in Culture for Molecular Functional and Imaging Studies. J Neurosci 36:3600–3610

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Miesenböck G, De Angelis DA, Rothman JE (1998) Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394:192–195

    Article  PubMed  Google Scholar 

  13. Gaffield MA, Betz WJ (2006) Imaging synaptic vesicle exocytosis and endocytosis with FM dyes. Nat Protoc 1:2916–2921

    Article  CAS  PubMed  Google Scholar 

  14. Kraszewski K, Mundigl O, Daniell L, Verderio C, Matteoli M, De Camilli P (1995) Synaptic vesicle dynamics in living cultured hippocampal neurons visualized with CY3-conjugated antibodies directed against the lumenal domain of synaptotagmin. J Neurosci 15:4328–4342

    CAS  PubMed  Google Scholar 

  15. Zhang Q, Cao Y, Tsien RW (2007) Quantum dots provide an optical signal specific to full collapse fusion of synaptic vesicles. Proc Natl Acad Sci U S A 104:17843–17848

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Zhang Q, Li Y, Tsien RW (2009) The dynamic control of kiss-and-run and vesicular reuse probed with single nanoparticles. Science 323:1448–1453

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Guillaud L, Dimitrov D, Takahashi T (2017) Presynaptic morphology and vesicular composition determine vesicle dynamics in mouse central synapses. eLife 6:e24845

    Google Scholar 

  18. Poo MM (2001) Neurotrophins as synaptic modulators. Nat Rev Neurosci 2:24–32

    Article  CAS  PubMed  Google Scholar 

  19. Yamashita T, Hige T, Takahashi T (2005) Vesicle endocytosis requires dynamin-dependent GTP hydrolysis at a fast CNS synapse. Science 307(5706):124–127

    Article  CAS  PubMed  Google Scholar 

  20. Ishikawa D, Takahashi N, Sasaki T, Usami A, Matsuki N, Ikegaya Y (2010) Fluorescent pipettes for optically targeted patch clamp recordings. Neural Netw 23:669–672

    Article  PubMed  Google Scholar 

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Author information

Authors and Affiliations

  1. Cellular and Molecular Synaptic Function Unit, Okinawa Institute of Science and Technology Graduate University, Okinawa, Japan

    Dimitar Dimitrov, Laurent Guillaud & Tomoyuki Takahashi

  2. Institute of Science and Technology Austria, Klosterneuburg, Austria

    Kohgaku Eguchi

Authors
  1. Dimitar Dimitrov
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  2. Laurent Guillaud
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  3. Kohgaku Eguchi
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  4. Tomoyuki Takahashi
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Corresponding author

Correspondence to Dimitar Dimitrov .

Editor information

Editors and Affiliations

  1. Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy

    Stephen D. Skaper

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Dimitrov, D., Guillaud, L., Eguchi, K., Takahashi, T. (2018). Culture of Mouse Giant Central Nervous System Synapses and Application for Imaging and Electrophysiological Analyses. In: Skaper, S. (eds) Neurotrophic Factors. Methods in Molecular Biology, vol 1727. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7571-6_15

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  • DOI: https://doi.org/10.1007/978-1-4939-7571-6_15

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7570-9

  • Online ISBN: 978-1-4939-7571-6

  • eBook Packages: Springer Protocols

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