Molecular assembly of auditory ribbon synapses in mice
by Dinah Burfeind
Date of Examination:2025-01-21
Date of issue:2025-01-17
Advisor:Prof. Dr. Tina, Pangršič
Referee:Prof. Dr. Tina Pangršič
Referee:Prof. Dr. Silvio Rizzoli
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Abstract
English
Auditory ribbon synapses are specialized structures in the inner hair cells (IHCs) that enable rapid neurotransmitter release, making them essential for precise signal transmission in the auditory system. However, insights into the developmental formation and maturation of these specialized synapses are still lacking. Investigating their composition provides crucial insights into their role in auditory development. This methodological doctoral thesis explores three approaches to studying the assembly of auditory ribbon structures. First, we analyzed the distribution patterns of mRNA of relevant proteins at the mRNA level using RNAscope. In this process, we modified the protocol so that, on one hand, we could outline cell bodies using immunohistochemistry, and on the other hand, examine whole cells through "floating samples." Next, we attempted to detect the in vivo translation of ribbon proteins using a puromycin-based Proximity Ligation Assay (PLA). While we successfully optimized puromycin uptake into the cells, the specificity of the PLA proved insufficient, limiting the applicability of this method in this context. Finally, we used isotopically labeled amino acids and NanoSIMS imaging to identify uptake in ribbon-associated regions. Here, we were able to show signs of increased uptake, which might indicate enhanced protein turnover during ribbon maturation. While RNAscope and NanoSIMS appeared as promising tools for studying ribbon formation, further validation with larger sample sizes is necessary to support these results. Ultimately, this research enhances our understanding of the molecular mechanisms driving ribbon synapse development and thereby providing valuable insights into auditory processing.
Keywords: NanoSIMS; Ribbon synapses; protein turnover; RNAscope; puromycin-based Proximity Ligation Assay (PLA)