Ultrastructural Imaging of Activity-Dependent Synaptic Membrane-Trafficking Events in Cultured Brain Slices

Research output: Contribution to journalJournal articleResearchpeer-review

  • Imig, Cordelia
  • Francisco José López-Murcia
  • Lydia Maus
  • Inés Hojas García-Plaza
  • Lena Sünke Mortensen
  • Manuela Schwark
  • Valentin Schwarze
  • Julie Angibaud
  • U Valentin Nägerl
  • Holger Taschenberger
  • Nils Brose
  • Benjamin H Cooper

Electron microscopy can resolve synapse ultrastructure with nanometer precision, but the capture of time-resolved, activity-dependent synaptic membrane-trafficking events has remained challenging, particularly in functionally distinct synapses in a tissue context. We present a method that combines optogenetic stimulation-coupled cryofixation ("flash-and-freeze") and electron microscopy to visualize membrane trafficking events and synapse-state-specific changes in presynaptic vesicle organization with high spatiotemporal resolution in synapses of cultured mouse brain tissue. With our experimental workflow, electrophysiological and "flash-and-freeze" electron microscopy experiments can be performed under identical conditions in artificial cerebrospinal fluid alone, without the addition of external cryoprotectants, which are otherwise needed to allow adequate tissue preservation upon freezing. Using this approach, we reveal depletion of docked vesicles and resolve compensatory membrane recycling events at individual presynaptic active zones at hippocampal mossy fiber synapses upon sustained stimulation.

Original languageEnglish
Issue number5
Pages (from-to)843-860
Number of pages18
Publication statusPublished - 2020

Bibliographical note

Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.

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