Molecular and structural investigation of assembly, maturation and heterogeneity of inner hair cell ribbon synapses
von Susann Michanski
Datum der mündl. Prüfung:2018-10-15
Betreuer:Prof. Dr. Tobias Moser
Gutachter:Prof. Dr. Tobias Moser
Gutachter:Prof. Dr. André Fiala
EnglischAge-related hearing and balance impairments represent the most common sensory deficits in humans. Both senses rely on hair cell ribbon synapses, which are highly specialized to sustain continuous release of neurotransmitter filled synaptic vesicles mediating indefatigable signal transmission. Disruption of their complex structural features are associated with disorders. However, their developmental aspects including the time course of ribbon synapse assembly, ribbon material targeting to active zones and their morphological maturation remain elusive. To address these questions, I first attempted to characterize the nanostructure of the auditory ribbon-type synapse by using electron microscopy and electron-tomography. The morphology of the inner hair cell (IHC) ribbon synapse was investigated in the mouse cochlea from late embryonic stages into adulthood. I could show that afferent synaptic contacts are established before floating spherical ribbon precursors arrive at immature active zones. Furthermore, pre-embedding immunogold labelings revealed two of the ribbon precursor constituents, namely RIBEYE and piccolino. The close proximity of floating ribbon precursors to cytoskeletal structures indicates active transport mechanisms to target them towards active zones. Moreover, ribbon fusion events around the onset of hearing serve as one key mechanism to transform multiple small ribbons per synaptic contact in IHCs of pre-hearing mice to a predominantly single and large synaptic ribbon in hearing animals. A similar structural confinement was found for the postsynaptic density. The employment of large 3D volume imaging techniques exhibited a morphological spatial gradient of active zones within individual IHCs, which is already established prior to hearing onset. Synapses of the modiolar side exhibited more frequently multiple ribbons per contact with generally larger sized ribbons, whereas pillar sides revealed a lower synapse density with contacts comprising a single and smaller ribbon. In the second part of this thesis, I studied the loss of the ribbon specific protein RIBEYE and its impact on the ultrastructural organization of cochlear IHC active zones. Multiple conventional-like active zones were present at each ribbonless synaptic contact of IHCs in RIBEYE knockout mice, which implies a partial compensation. Exploring older mature animals excluded a developmental delay in these knockout mice. In the third part, I compared my developmental results from auditory IHCs with the two different types of vestibular hair cells from the utricle. While developmental processes have been studied extensively in the auditory pathway, much less is reported for the vestibular system. Utricular hair cells can be divided into type I and type II cells. In contrast to cochlear IHCs, they do not exhibit a change in size or number of synaptic ribbons per synapse or in the number of synaptic vesicles upon maturation. Unexpectedly, floating ribbons were present even in 11 months old mice arguing against a pure precursor function as described for immature IHCs. The number of floating ribbons increased during development in type I cells forming clusters, but decreased in type II cells. I propose that the large number of floating ribbons in type I hair cell could be the result of a secondary detachment of synaptic ribbons, which indicates a distinct maturation compared to type II and cochlear HCs. Type II hair cells showed a sequence of maturational events, which was more comparable to cochlear IHCs. In conclusion, mapping hair cell synapses during development resulted in the discovery of structural modifications, which correlate to functional maturation processes of the active zone. If also occurring in mature hair cells, the observed fusion or detachment events of ribbon material might represent a mechanism to modulate the ribbon size and number, which might influence the respective synaptic strength in cochlear and vestibular hair cells.
Keywords: Inner hair cell, ribbon synapse, maturation, heterogeneity, cochlea, utricle, high resolution microscopy, electron microscopy