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.
Highlights
Communication across chemical synapses relies on the release of neurotransmitter from synaptic vesicles, through vesicle fusion to the presynaptic plasma membrane
We explored here the possibility of studying membrane trafficking in inner hair cells of the auditory pathway (IHCs) using the technique of photo-oxidation electron microscopy, in which the FM dye taken up by the organelles is converted to a dark precipitate that is visible in electron microscopy
As indicated in the Introduction, it is still debated whether FM dyes can be used as reliable reporters of endocytosis in IHCs
Summary
Communication across chemical synapses relies on the release of neurotransmitter from synaptic vesicles, through vesicle fusion to the presynaptic plasma membrane (exocytosis). Studies focusing on conventional synapses from both the central and peripheral nervous systems have revealed several different mechanisms of vesicle recycling. One such mechanism is kiss-and-run, in which the fusion pore of the vesicle closes rapidly after exocytosis. The fused vesicle may be retrieved by clathrin-mediated endocytosis (CME), either directly from the plasma membrane, or from membrane infoldings that form on the surface of the synapse after abundant exocytosis. The latter mechanism is known as bulk endocytosis [1,2]
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