Optogenetics and electron tomography for structure-function analysis of cochlear ribbon synapses
- University of Göttingen, Germany
- Max Planck Institute for Multidisciplinary Sciences, Germany
- University Medical Center Göttingen, Germany
- South Westphalia University of Applied Sciences, Germany
- University of Göttingen Medical Center, Germany
Abstract
Ribbon synapses of cochlear inner hair cells (IHCs) are specialized to indefatigably transmit sound information at high rates. To understand the underlying mechanisms, structure-function analysis of the active zone (AZ) of these synapses is essential. Previous electron microscopy studies of synaptic vesicle (SV) dynamics at the IHC AZ used potassium stimulation, which limited the temporal resolution to minutes. Here, we established optogenetic IHC stimulation followed by quick freezing within milliseconds and electron tomography to study the ultrastructure of functional synapse states with good temporal resolution in mice. We characterized optogenetic IHC stimulation by patch-clamp recordings from IHCs and postsynaptic boutons revealing robust IHC depolarization and transmitter release. Ultrastructurally, the number of docked SVs increased upon short (17-25 ms) and long (48-76 ms) light stimulation paradigms. We did not observe enlarged SVs or other morphological correlates of homotypic fusion events. Our results indicate a rapid recruitment of SVs to the docked state upon stimulation and suggest that univesicular release prevails as the quantal mechanism of exocytosis at IHC ribbon synapses.
Data availability
All research materials and biological reagents used in this paper are reported in the Materials and Method section. The custom routines and scripts used in the manuscript are provided as Source Codes: Source Code 1: IMARIS custom plug-ins for the analysis of Figure 1D; Source code 2: Igor Pro custom-written analysis (OptoEPSCs) of light-evoked EPSCs related to Figure 3C-F; Source code 3: MATLAB scripts (HPMacquire) for the computer interface to control the light pulse for Opto-HPF. Related to Figure 4A; Source code 4: MATLAB script (Intensityprofilecalculator) for the analysis of the irradiance in Figure 4E; Source code 5: MATLAB scripts (HPManalyse) for the alignment of the data obtained from the Opto-HPF sensors. Related to Figure 5C. The raw data files, including the numerical data associated with the figures, are available on the Open Science Framework DOI 10.17605/OSF.IO/WFJVE .
Article and author information
Author details
Lina María Jaime Tobón
Funding
Deutsche Forschungsgemeinschaft (CRC 889,Project A02)
- Tobias Moser
Multiscale Bioimaging is a Cluster of Excellence of the University of Göttingen, Germany (EXC 2067/1- 390729940)
- Tobias Moser
MPI-NAT (Erwin Neher Fellowship)
- Lina María Jaime Tobón
Deutsche Forschungsgemeinschaft (CRC 889,Project A07)
- Carolin Wichmann
Deutsche Forschungsgemeinschaft (CRC 1286,Project A04)
- Carolin Wichmann
Deutsche Forschungsgemeinschaft (CRC 1286,Project B05)
- Tobias Moser
Deutsche Forschungsgemeinschaft (CRC 1286,Project Z04)
- Felipe Opazo
Leibniz Program (Leibniz Prize)
- Tobias Moser
Niedersächsisches Ministerium für Wissenschaft und Kultur (Niedersächsisches Vorab)
- Tobias Moser
Erasmus Mundus (Neurasmus Scholarship)
- Lina María Jaime Tobón
Fondation Pour l'Audition (FPA RD-2020-10)
- Tobias Moser
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: Animal handling and all experimental procedures were in accordance with the national animal care guidelines issued by the animal welfare committees of the University of Göttingen and the Animal Welfare Office of the State of Lower Saxony (AZ 509.42502/01-27.03).
Reviewing Editor
- Andrew J King, University of Oxford, United Kingdom
Publication history
- Received: April 14, 2022
- Preprint posted: May 10, 2022 (view preprint)
- Accepted: November 28, 2022
- Accepted Manuscript published: December 23, 2022 (version 1)
Copyright
© 2022, Chakrabarti et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Optogenetics and electron tomography for structure-function analysis of cochlear ribbon synapses
eLife 11:e79494.
https://doi.org/10.7554/eLife.79494
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