Ultrastructural study of synaptic vesicle dynamics in murine inner hair cell ribbon synapses under sustained stimulation
by Magdalena Redondo Canales
Date of Examination:2023-04-21
Date of issue:2023-07-11
Advisor:Prof. Dr. Carolin Wichmann
Referee:Prof. Dr. Tim Gollisch
Referee:Prof. Dr. Nils Brose
Referee:Prof. Dr. Rubén Fernández-Busnadiego
Referee:Prof. Dr. Christine Stadelmann-Nessler
Referee:Prof. Dr. Silvio O. Rizzoli
Persistent Address:
http://dx.doi.org/10.53846/goediss-9984
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Abstract
English
Transmission of auditory information relies in the correct functioning of inner hair cell (IHC) rib-bon synapses, which mediate synaptic vesicle release in an indefatigable manner. The name-giving synaptic ribbon and the formation of tethers at the active zone membrane are key to maintain coordinated and sustained exocytosis. Furthermore, endocytosis, which has been iden-tified to happen as clathrin-mediated and bulk endocytosis, is essential to retrieve material upon prolonged stimulation. This thesis focuses on the study of the ribbon synapse ultrastructure upon sustained stimulation to elucidate the role of otoferlin (Otof) and RIBEYE (RBE) in the syn-aptic vesicle cycle. On the one hand, Otof is a protein essential for exocytosis, where it has been proposed to be a tether component, as well as being suggested to be involved in endocytosis. On the other hand, RBE is the most abundant constituent of the synaptic ribbon, whose disrup-tion results in ribbon-less synapses and a mild impairment of the ribbon synapse function. Hence, the availability of synaptic vesicles and a fully functional vesicle cycle is required for nor-mal ribbon synapse function. Therefore, the vesicle dynamics in Otof and RBE mutant ribbon synapses was studied via strong and prolonged chemical stimulation followed by high-pressure freezing and electron tomography. Vesicles were classified into different pools, according to their position within the ribbon synapse, as well as vesicle subpools, according to the presence of tethers that are thought to mediate vesicle recruitment for release. In addition, precise milli-second stimulation of IHCs has been recently achieved via optogenetics by expressing chan-nelrhodopsin 2 (ChR2) in IHCs, allowing to observe the early stages of exocytosis. Therefore, I investigated the contribution of Ca2+ influx to exocytosis passing through ChR2 upon prolonged stimulation. In this regard, I established a 500 ms light stimulation paradigm, which was followed by high-pressure freezing and electron tomography, where the different vesicle pools and sub-pools were analysed. The role of Otof as an actor in exocytosis and endocytosis was investigated using a 15-minute, chemical stimulation paradigm and tomogram analysis. Morphological correlates of exocytosis and endocytosis were analysed and compared in two Otof mutants, namely Otof knockout (KO) and pachanga (Otofpga/pga) to better propose a role of Otof in the vesicle cycle. Vesicle subpool analysis revealed that the presence of Otof is necessary for vesicles to establish initial tethering with the active zone membrane. Conversely, endocytic correlate data make Otof remain unclear, since more endocytic structures were found in resting OtofKO/KO ribbon synapses compared to stimulated OtofKO/KO, as well as resting wild-type and Otofpga/pga synapses. This vesicle clusters might indicate an impairment in the endocytic pathway. Furthermore, clusters of large vesicles were observed in some mutant ribbon synapses, although their molecular identity remains un-known. Overall, these data indicate Otof to be essential in the approach of tethered vesicles upon prolonged stimulation to the plasma membrane until docking. Second, the role of the synaptic ribbon was studied using RBEKO/KO IHCs, which resulted ribbon-less active zones. I used a 15-minute chemical stimulation paradigm and followed by tomogram analysis to compared stimulated and inhibited wild-type and mutant samples. Vesicle subpool analysis showed that the synaptic ribbon seems to be involved in the approach of vesicles to the active zone membrane. Furthermore, lack of synaptic ribbon appears to affect the topography of docked vesicles laterally to the active zone membrane, which might be indicative of an alter-ation in the coupling of Ca2+influx-to-vesicle. Importantly, due to the partial dataset is not pos-sible to draw any final conclusion from this work. In summary, the preliminary ultrastructural data suggest that the ribbon might be a key structure modulating the approach of vesicles to the plasma membrane and their position prior to release. Third, the contribution of ChR2 in Ca2+ influx for exocytosis in optogenetically stimulable IHCs was assessed applying a 500 ms light stimulation paradigm and subsequent vesicle subpool anal-ysis. To this end, blockage of IHC constitutive Ca2+ channels was performed using isradipine in stimulatory conditions, which was compared to inhibited and stimulated (in the absence of is-radipine) conditions. No significant differences were found here, and future experiments will need to be done to confirm the preliminary observations. However, my study revealed long term ChR2 expression, which did not affect the synaptic progression along adulthood. In conclusion, the study of the morphological correlates of exocytosis and endocytosis upon pro-longed stimulation suggest that docked vesicles greatly depend on the presence of Otof to reach such state, and the synaptic ribbon to localise them normally along the active zone membrane. Completion of the study on Ca2+ influx through ChR2, as well as the creation of optogenetically stimulable IHCs lacking Otof or the synaptic ribbon will allow to obtain temporally precise ultra-structural information of the synaptic vesicle cycle.
Keywords: Inner hair cells; Ribbon synapses; Otoferlin; RIBEYE; High-pressure freezing; Vesicle cycle