Optogenetics and electron tomography for structure-function analysis of cochlear ribbon synapses

  1. Rituparna Chakrabarti
  2. Lina María Jaime Tobón
  3. Loujin Slitin
  4. Magdalena Redondo-Canales
  5. Gerhard Hoch
  6. Marina Slashcheva
  7. Elisabeth Fritsch
  8. Kai Bodensiek
  9. Özge Demet Özçete
  10. Mehmet Gültas
  11. Susann Michanski
  12. Felipe Opazo
  13. Jakob Neef
  14. Tina Pangrsic
  15. Tobias Moser  Is a corresponding author
  16. Carolin Wichmann  Is a corresponding author
  1. University of Göttingen, Germany
  2. Max Planck Institute for Multidisciplinary Sciences, Germany
  3. University Medical Center Göttingen, Germany
  4. South Westphalia University of Applied Sciences, Germany
  5. 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 .

The following data sets were generated

Article and author information

Author details

  1. Rituparna Chakrabarti

    Collaborative Research Center 889, University of Göttingen, Göttingen, Germany
    Competing interests
    No competing interests declared.
  2. Lina María Jaime Tobón

    Auditory Neuroscience and Synaptic Nanophysiology Group, Max Planck Institute for Multidisciplinary Sciences, Göttingen, Germany
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6752-7750
  3. Loujin Slitin

    Molecular Architecture of Synapses Group, University Medical Center Göttingen, Göttingen, Germany
    Competing interests
    No competing interests declared.
  4. Magdalena Redondo-Canales

    Molecular Architecture of Synapses Group, University Medical Center Göttingen, Göttingen, Germany
    Competing interests
    No competing interests declared.
  5. Gerhard Hoch

    Institute for Auditory Neuroscience, University Medical Center Göttingen, Göttingen, Germany
    Competing interests
    No competing interests declared.
  6. Marina Slashcheva

    Göttingen Graduate School for Neuroscience and Molecular Biosciences, University of Göttingen, Göttingen, Germany
    Competing interests
    No competing interests declared.
  7. Elisabeth Fritsch

    Göttingen Graduate School for Neuroscience and Molecular Biosciences, University of Göttingen, Göttingen, Germany
    Competing interests
    No competing interests declared.
  8. Kai Bodensiek

    Institute for Auditory Neuroscience, University Medical Center Göttingen, Göttingen, Germany
    Competing interests
    No competing interests declared.
  9. Özge Demet Özçete

    Collaborative Research Center 889, University of Göttingen, Göttingen, Germany
    Competing interests
    No competing interests declared.
  10. Mehmet Gültas

    Faculty of Agriculture, South Westphalia University of Applied Sciences, Soest, Germany
    Competing interests
    No competing interests declared.
  11. Susann Michanski

    Molecular Architecture of Synapses Group, University Medical Center Göttingen, Göttingen, Germany
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5893-1981
  12. Felipe Opazo

    Center for Biostructural Imaging of Neurodegeneration, University of Göttingen Medical Center, Goettingen, Germany
    Competing interests
    Felipe Opazo, Felipe Opazo is a shareholder of Nanotag Biotechnologies GmbH..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4968-9713
  13. Jakob Neef

    Institute for Auditory Neuroscience, University Medical Center Göttingen, Göttingen, Germany
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4757-9385
  14. Tina Pangrsic

    Institute for Auditory Neuroscience, University Medical Center Göttingen, Göttingen, Germany
    Competing interests
    No competing interests declared.
  15. Tobias Moser

    Department of Otolaryngology, University of Göttingen, Göttingen, Germany
    For correspondence
    tmoser@gwdg.de
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7145-0533
  16. Carolin Wichmann

    Molecular Architecture of Synapses Group, University Medical Center Göttingen, Göttingen, Germany
    For correspondence
    Carolin.Wichmann@medizin.uni-goettingen.de
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8868-8716

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

  1. Andrew J King, University of Oxford, United Kingdom

Version history

  1. Received: April 14, 2022
  2. Preprint posted: May 10, 2022 (view preprint)
  3. Accepted: November 28, 2022
  4. Accepted Manuscript published: December 23, 2022 (version 1)
  5. Version of Record published: February 8, 2023 (version 2)

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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  1. Rituparna Chakrabarti
  2. Lina María Jaime Tobón
  3. Loujin Slitin
  4. Magdalena Redondo-Canales
  5. Gerhard Hoch
  6. Marina Slashcheva
  7. Elisabeth Fritsch
  8. Kai Bodensiek
  9. Özge Demet Özçete
  10. Mehmet Gültas
  11. Susann Michanski
  12. Felipe Opazo
  13. Jakob Neef
  14. Tina Pangrsic
  15. Tobias Moser
  16. Carolin Wichmann
(2022)
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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