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

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 o...

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Bibliographic Details
Published in:Neuron (Cambridge, Mass.) Vol. 108; no. 5; p. 843
Main Authors: Imig, Cordelia, López-Murcia, Francisco José, Maus, Lydia, García-Plaza, Inés Hojas, Mortensen, Lena Sünke, Schwark, Manuela, Schwarze, Valentin, Angibaud, Julie, Nägerl, U Valentin, Taschenberger, Holger, Brose, Nils, Cooper, Benjamin H
Format: Journal Article
Language:English
Published: United States 09.12.2020
Subjects:
Animals
Excitatory Postsynaptic Potentials - physiology
Gene Knock-In Techniques - methods
Hippocampus - physiology
Hippocampus - ultrastructure
Mice
Mice, Transgenic
Microscopy, Electron - methods
Microtomy - methods
Organ Culture Techniques
Protein Transport - physiology
Synaptic Membranes - physiology
Synaptic Membranes - ultrastructure
High-pressure freezing
active zone
optogenetics
Electron tomography
endocytosis
exocytosis
synapse
Electron microscopy
Flash-and-freeze
synaptic vesicle
ISSN:1097-4199, 1097-4199
Online Access:Get more information
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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.
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ISSN:1097-4199
1097-4199
DOI:10.1016/j.neuron.2020.09.004