Investigating the Ultrastructural Consequences of Aging on Murine Inner Hair Cell Ribbon Synapses
by Loujin Slitin
Date of Examination:2023-06-29
Date of issue:2024-03-05
Advisor:Prof. Dr. Carolin Wichmann
Referee:Prof. Dr. Tiago Outeiro
Referee:Dr. Wiebke Möbius
Persistent Address:
http://dx.doi.org/10.53846/goediss-10385
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Abstract
English
The ribbon synapses between inner hair cells (IHCs) and spiral ganglion neurons (SGNs) are first synapses of the auditory pathway. They mediate the harmonized and tireless neurotransmission needed for continuous sound encoding. Additionally, these synapses display an unconventional molecular architecture well-suited to sustain high rates and exceptional temporal precision in signal transduction. Synaptic vesicles (SVs) at the ribbon synapse are organized, according to their location, in morphologically distinct pools; the ribbon-associated (RA) and the membrane-proximal (MP) pool. Proteinaceous filaments bind these SVs to different structures (i.g. the ribbon and the active zone (AZ) membrane) and thus, by considering the number and location to which these tethers bind, the concept of SVs sub-pools was proposed to indicate different functional stages of exocytosis. In order to unravel the dynamics of these (sub)pools, we employ a temporally-resolved structure-function analysis using IHCs optogenetic stimulation in a millisecond range followed by high pressure freezing (HPF). After subsequent freeze-substitution (FS) we employed electron tomography (ET) to visualize fine-structural changes like tethers and SV docking. We demonstrate a rapid recruitment of SVs to the morphologically docked state upon stimulation. Morphological correlates of homotypic fusion events could not be observed under our stimulation paradigms. Therefore, we suggest the at IHC ribbon synapses, univesicular mode of release to prevails as the release mechanism of exocytosis. Accumulating evidence in aging research point out ribbon synapses among the highly vulnerable and early affected structures upon aging leading to age-related cochlear synaptopathy. It remains elusive, when and how precisely these synapses degenerate with age. Therefore, I employed HPF/FS and ET to assess the consequences of aging of ribbon presynaptic AZs on the ultrastructural level. Using two distinct wildtypes (WT): CBA/J (CBAJ), as normally aging WT and C57BL/6J (B6J), a WT with an early-onset of progressive hearing loss. Furthermore, I addressed the aging in two, very essential knock-out (KO) models, (i) the congenitally deaf otoferlin KO (OtofKO/KO) mice, and the RIBEYE KO (RBEKO/KO) mice that show ribbon-less AZs. In CBAJ, B6J and OtofKO/KO, the comparable numbers of the MP- and RA-SVs upon aging indicated that the synaptic ribbons were still capable of promoting a complementary of SVs in both pools. In 6 weeks old CBAJ AZs, docked SVs were not as rare as previously reported for B6J mice but they significantly decreased upon aging. However, the distribution of SVs was altered upon aging in all genotypes, though partially with opposite effects. Older synapses of CBAJ and RBEKO/KO demonstrated significantly increased distances of SVs from the AZ membrane compared to younger ones. Interestingly, OtofKO/KO AZs were the only ones to reveal the opposite observation of shorter SV distances from the AZ membrane upon aging along with increased number of tethered SVs at the expense of non-tethered SVs. Considering otoferlin’s potential role in replenishment of release ready SVs and release site clearance, the loss of otoferlin might have led to SVs accumulation in close proximity to the AZ membrane and thus, masked the age-induced alteration in SVs distribution. Using focused ion beam-scanning electron microscopy (FIB-SEM) to visualize the basal compartment of B6J (1y8m) and OtofKO/KO (1y5m) IHCs, I found a reduction of SGNs and ribbon-occupied synaptic contacts. The remaining SGN boutons were located at the most basal part of the IHCs. Moreover, OtofKO/KO IHCs exhibited a preferential modiolar ribbon loss. In summary, my thesis gives insight into activity-dependent changes of ribbon synapse structure and reveals ultrastructural changes on the level of SV pools in B6J animals already prior to onset of early progressive hearing loss.
Keywords: Aging; ribbon synapses; otoferlin; RIBEYE; electron tomography; high pressure freezing; FIB-SEM
