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PLoS One. 2014 Feb 5;9(2):e88353. doi: 10.1371/journal.pone.0088353. eCollection 2014.

FM dye photo-oxidation as a tool for monitoring membrane recycling in inner hair cells.

Author information

1
Department of Neuro- and Sensory Physiology, University Medical Center Göttingen, Göttingen, Germany ; STED Microscopy of Synaptic Function, European Neuroscience Institute, Göttingen, Germany.
2
Department of Neuro- and Sensory Physiology, University Medical Center Göttingen, Göttingen, Germany ; STED Microscopy of Synaptic Function, European Neuroscience Institute, Göttingen, Germany ; International Max Planck Research School Neurosciences, Göttingen, Germany ; Collaborative Research Center 889, University of Göttingen, Göttingen, Germany.
3
Department of Neuro- and Sensory Physiology, University Medical Center Göttingen, Göttingen, Germany ; Collaborative Research Center 889, University of Göttingen, Göttingen, Germany ; Cluster of Excellence Nanoscale Microscopy and Molecular Physiology of the Brain, University of Göttingen, Göttingen, Germany.

Abstract

Styryl (FM) dyes have been used for more than two decades to investigate exo- and endocytosis in conventional synapses. However, they are difficult to use in the inner hair cells of the auditory pathway (IHCs), as FM dyes appear to penetrate through mechanotransducer channels into the cytosol of IHCs, masking endocytotic uptake. To solve this problem we applied to IHCs the FM dye photo-oxidation technique, which renders the dyes into electron microscopy markers. Photo-oxidation allowed the unambiguous identification of labeled organelles, despite the presence of FM dye in the cytosol. This enabled us to describe the morphologies of several organelles that take up membrane in IHCs, both at rest and during stimulation. At rest, endosome-like organelles were detected in the region of the cuticular plate. Larger tubulo-cisternal organelles dominated the top and nuclear regions. Finally, the basal region, where the IHC active zones are located, contained few labeled organelles. Stimulation increased significantly membrane trafficking in the basal region, inducing the appearance of labeled vesicles and cistern-like organelles. The latter were replaced by small, synaptic-like vesicles during recovery after stimulation. In contrast, no changes in membrane trafficking were induced by stimulation in the cuticular plate region or in the top and nuclear regions. We conclude that synaptic vesicle recycling takes place mostly in the basal region of the IHCs. Other organelles participate in abundant constitutive membrane trafficking throughout the rest of the IHC volume.

PMID:
24505482
PMCID:
PMC3914975
DOI:
10.1371/journal.pone.0088353
[Indexed for MEDLINE]
Free PMC Article

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