Ultrastructural Imaging of Activity-Dependent Synaptic Membrane-Trafficking Events in Cultured Brain Slices
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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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The mossy fiber-CA3 PN synapse is accessible to quantitative structural analysis by electron microscopy, high-pressure freezing, and freeze-fracture replica labeling.
“Finally, it is accessible to quantitative structural analysis by EM [34,35], high-pressure freezing (HPF) [36–38], and freeze-fracture replica labeling (FRL) [39].”
From Presynaptic cAMP-PKA-mediated potentiation induces reconfiguration of synaptic vesicle pools and channel-vesicle coupling at hippocampal mossy fiber boutons (2024) · cites this paper as [38]
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“Kiss-shrink-run” unifies mechanisms for synaptic vesicle exocytosis and hyperfast recycling
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Automated Detection and Localization of Synaptic Vesicles in Electron Microscopy Images
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Fully-primed slowly-recovering vesicles mediate presynaptic LTP at neocortical neurons
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FMRP Sustains Presynaptic Function via Control of Activity-Dependent Bulk Endocytosis
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SynapseNet: Deep learning for automatic synapse reconstruction
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“Finally, it is accessible to quantitative structural analysis by EM [34,35], high-pressure freezing (HPF) [36–38],and freeze-fracture replica labeling (FRL) [39].”
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Ultrastructural analysis of wild-type and RIM1α knockout active zones in a large cortical synapse
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Endocytic proteins: An expanding repertoire of presynaptic functions
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Mechanistic insights into cAMP-mediated presynaptic potentiation at hippocampal mossy fiber synapses
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The synaptic vesicle cluster as a controller of pre‐ and postsynaptic structure and function
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Self-citationCurrent molecular approaches to investigate pre‐synaptic dysfunction
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Structural insights into lipid membrane binding by human ferlins
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The molecular infrastructure of glutamatergic synapses in the mammalian forebrainPreprint
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Methods for multiscale structural and functional analysis of the mammalian cochlea
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Cav2.2 Channels Sustain Vesicle Recruitment at a Mature Glutamatergic Synapse
Wender, M., Bornschein, G., Brachtendorf, S., Hallermann, S., Eilers, J., & Schmidt, H. (2023) · Journal of Neuroscience · doi:10.1523/jneurosci.1279-22.2023 · Cited by 6 · see citing works
Dynamic nanoscale architecture of synaptic vesicle fusion in mouse hippocampal neurons
Kroll, J., Kravčenko, U., Sadeghi, M., Diebolder, C. A., Ivanov, L., Lubas, M., Sprink, T., Schacherl, M., Kudryashev, M., & Rosenmund, C. (2025) · Nature Communications · doi:10.1038/s41467-025-67291-6 · Cited by 5 · see citing works
Time Resolved Cryo‐Correlative Light and Electron Microscopy
Szabo, G. V., & Burg, T. P. (2024) · Advanced Functional Materials · doi:10.1002/adfm.202313705 · Cited by 5 · see citing works
Höltje, M., Wolkowicz, A., Brunk, I., Baron, J., & Ahnert‐Hilger, G. (2024) · Journal of Neurochemistry · doi:10.1111/jnc.16248 · Cited by 4 · see citing works
Visualizing Presynaptic Active Zones and Synaptic Vesicles
Heckmann, M., & Pauli, M. (2022) · Frontiers in Synaptic Neuroscience · doi:10.3389/fnsyn.2022.901341 · Cited by 3 · see citing works
Ultrastructural membrane dynamics of mouse and human cortical synapses
Eddings, C. R., Fan, M., Imoto, Y., Itoh, K., McDonald, X., Eilers, J., Anderson, W. S., Worley, P. F., Lippmann, K., Nauen, D. W., et al. (2025) · Neuron · doi:10.1016/j.neuron.2025.10.030 · Cited by 3 · see citing works
The molecular infrastructure of glutamatergic synapses in the mammalian forebrainPreprint
Peukes, J., Lovatt, C., Leistner, C., Jérôme Boulanger, A., Morado, D. R., Fuller, M. J., Kukulski, W., Zhu, F., Komiyama, N. H., Briggs, J. A. G., et al. (2024) · eLife · doi:10.7554/elife.100335 · Cited by 3 · see citing works
The molecular infrastructure of glutamatergic synapses in the mammalian forebrain
Peukes, J., Lovatt, C., Leistner, C., Jérôme Boulanger, A., Morado, D. R., Fuller, M. J., Kukulski, W., Zhu, F., Komiyama, N. H., Briggs, J. A. G., et al. (2025) · eLife · doi:10.7554/elife.100335.3 · Cited by 2 · see citing works
Memory engram synapse 3D macromolecular architecture visualized by cryoCLEM-guided cryoET
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Subcellular depletion of importin β1 impairs presynaptic local translation and spatial memory
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Neddylation regulates the development and function of glutamatergic neurons
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The flexible synapse – How mossy fiber architecture adapts to changing needs
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Encephalopathy-linked UFM1 variants impede neuronal protein translation, development, and function
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Self-citationDigging Deeper: Advancements in Visualization of Inhibitory Synapses in Neurodegenerative Disorders
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Distinguishing between Synaptic Vesicles in Different Functional States
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Mitochondrial adaptation at the neuronal presynapse
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Electron Tomographic Methods for Studying Organelles of the Murine Chemical Synapse
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Molecular neuroscience community shares perspectives
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Engineered proteins to restore and enhance neurotransmitter releasePreprint
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- Authors
- Imig, C., López-Murcia, F. J., Maus, L., García-Plaza, I. H., Mortensen, L. S., Schwark, M., Schwarze, V., Angibaud, J., Nägerl, U. V., Taschenberger, H., Brose, N., & Cooper, B. H.
- Affiliations
- Max Planck Institute of Experimental Medicine (DE)
- Centre National de la Recherche Scientifique (FR)
- Corresponding author
- Imig, Cordelia
- Brose, Nils
- Cooper, Benjamin H.
- ORCID
- Venue
- Neuron
- Publisher
- Elsevier BV
- Published
- 2020-12
- Volume
- 108
- Issue
- 5
- Pages
- 843-860.e8
- ISSN
- 0896-6273 (ISSN-L)
- PMC
- PMC7736621
- OpenAlex
- W3088769972
- Cited by
- 66
- References
- 85
- Citations by year
- 2026: 6 · 2025: 11 · 2024: 10 · 2023: 13 · 2022: 14 · 2021: 11 · 2020: 1
- FWCI
- 4.47
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Neuron (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Research at the University of Copenhagen (University of Copenhagen) (publishedVersion, cc-by)
- Dipòsit Digital de la Universitat de Barcelona (Universitat de Barcelona) (submittedVersion, cc-by-nc-nd)
- GoeScholar The Publication Server of the Georg-August-Universität Göttingen (Georg-August-Universität Göttingen) (submittedVersion, cc-by)
- MPG.PuRe (Max Planck Society) (submittedVersion, other-oa)
- MPG.PuRe (Max Planck Society) (submittedVersion)
- Funders
- European Commission (ERC Advanced Grant SynPrime)
- Deutsche Forschungsgemeinschaft (SFB1286/A01)
- Germany’s Excellence Strategy (EXC 2067/1-390729940)
- IMPRS Neurosciences
- Topics
- Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Advanced Electron Microscopy Techniques and Applications
- Keywords
- presynaptic vesicles, optogenetic stimulation, cryofixation, active zone, docked vesicles, membrane recycling, synaptic ultrastructure
- MeSH terms
- Animals, Hippocampus, Mice, Transgenic, Microscopy, Electron, Microtomy, Organ Culture Techniques, Synaptic Membranes, Excitatory Postsynaptic Potentials, Protein Transport, Mice, Gene Knock-In Techniques
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Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
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