| dc.description.abstracteng | The inner hair cell ribbon synapses are the relay stations of the auditory pathway, attuned to transmit acoustic information at rates of up to hundreds of Hz. Such indefatigable signal transmission demands ultrafast vesicle replenishment mechanisms, which ensure a constant resupply of vesicles to the active zone. At these high-throughput synapses, the synaptic ribbon provides a molecular scaffold to orchestrate the spatial localization of synaptic vesicles. However, the molecular mechanism(s) coordinating vesicular release remains elusive. This thesis aims to correlate functional deficits of inner hair cell synaptic transmission with ultrastructural characteristics. Using advanced electron microscopy, snapshots of vesicle pools were captured under different stimulation paradigms, including short light pulses as well as mild and strong K+ stimulation to visualize early vesicular release and sustained exocytosis respectively. The images are then compiled into a montage to understand the vesicle dynamics in inner hair cells morphologically. Moreover, to decipher the molecular players maintaining vesicle pools at the active zone, different point and/or deletion mouse mutants of otoferlin – a key regulator for the last stages of exocytosis in inner hair cells – and the presynaptic scaffold Rab-3 interacting molecule (RIM) 2α were investigated ultrastructurally.
Firstly, using hearing impaired mice carrying a temperature-sensitive mutation in the C2C domain of otoferlin (OtofI515T/I515T), I could show that otoferlin is involved in vesicle reformation. Here, coinciding with a vesicle replenishment deficit obtained by inner hair cell capacitance measurements, the ultrastructural phenotype was dominated by enlarged vesicle diameters.
Secondly, I focused on inter-structural connectivity by proteinaceous filaments. Here, two types of connections were characterized: (i) inter-connectors, which are formed between vesicles or (ii) tethers, linking vesicles to other synaptic structures such as the ribbon and/or the presynaptic membrane. A closer analysis of these structures showed a higher complexity of connections at the distal end of the ribbon upon stimulation. However, closer to the active zone the multiple-tethered and docked vesicles were rare. Using a replenishment-impaired otoferlin pachanga mutant (OtofPga/Pga) allowed me to study the role of these filaments in vesicle replenishment. OtofPga/Pga mice exhibit reduced filament connectivity, with compactly packed vesicles around the ribbon and accumulation of multiple-tethered and docked vesicles at the membrane. This suggested a role of otoferlin downstream of docking, such as vesicular fusion or subsequent release site clearance. Impairment of either one of these processes may result in the accumulation of release-ready vesicles at the membrane, which in turn creates a roadblock for the vesicles at the ribbon. Finally, to find other potential vesicle tethering candidates at inner hair cells, RIM2α was investigated. RIM2α deficient inner hair cells revealed a reduced fraction of tethered vesicles at the membrane and proximal part of the ribbon (closer to the active zone), supporting the role of RIM2α in vesicle tethering.
In the end, to obtain ultrafast immobilization, immediately after stimulation of vesicular release, an inner hair cell-specific channelrhodopsin-2 knock-in mouse model was used. Here, short light pulses prior to high-pressure freezing showed a fast refilling of the membrane-proximal vesicle pool, while ribbon-associated vesicles were reduced under these conditions. Therefore, optogenetic stimulation proved to be a useful tool to understand the structure-function relationship of inner hair cells. However, this experimental paradigm still requires further optimization to capture early exocytosis events, for example, vesicle fusion events. | de |