Neuron
Volume 108, Issue 5, 9 December 2020, Pages 843-860.e8
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Ultrastructural Imaging of Activity-Dependent Synaptic Membrane-Trafficking Events in Cultured Brain Slices

https://doi.org/10.1016/j.neuron.2020.09.004Get rights and content
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Highlights

  • High-pressure freezing of cultured brain tissue without cryoprotectants for EM

  • Flash-and-freeze functional EM of identified synapses in complex neural circuits

  • Direct correlation of activity-induced functional states and ultrastructure

  • Dissection of the spatiotemporal organization of endocytosis in identified synapses

Summary

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.

Keywords

synapse
synaptic vesicle
active zone
exocytosis
endocytosis
Electron microscopy
Electron tomography
optogenetics
High-pressure freezing
Flash-and-freeze

Cited by (0)

6

Present address: Department of Neuroscience, University of Copenhagen, 2200 Copenhagen, Denmark

7

Present address: Interdisciplinary Center for Bioinformatics, University of Leipzig, 0417 Leipzig, Germany

8

Lead Contact