The molecular anatomy of synaptic vesicle recycling at the hair cell ribbon synapse

Abstract

The inner hair cell (IHC) ribbon synapse of the auditory system is a highly specialized synapse, adapted to very fast transmission rates and maintenance of synaptic vesicle release over long periods of time without showing substantial fatigue. These properties make the IHC ribbon synapse an interesting target to study synaptic vesicle exo- and endocytosis, since there is a need for very efficient synaptic vesicle recycling, in order to ensure vesicle replenishment during prolonged exocytosis, with high release rates. Current knowledge about the function of these synapses mainly stems from electrophysiological measurements, focusing on release kinetics, Ca2+ channel distribution and vesicle pool sizes. However, a detailed picture of the molecular organization of the IHC ribbon synapse, and especially of the proteins involved in the synaptic vesicle recycling process, is still missing. This is due to major difficulties in studying synaptic proteins in the IHC using fluorescence imaging techniques, since immunostaining protocols using commercially-available antibodies often provide insufficient staining quality for IHCs. A better understanding of how the IHC ribbon synapse is organized would facilitate research on functional processes by relating its structure to its function. Therefore, in this work I set out to improve the methods for the investigation of IHC ribbon synapses by immunofluorescence microscopy. I have established glyoxal as an alternative fixative to PFA. Furthermore, I have developed a method (CosiQuant) to estimate protein copy numbers using a comparative imaging approach. CosiQuant is based on the comparison of immunostaining signals between a sample of interest and biochemically-characterized synaptosomes with known protein copy numbers. This method is particularly useful for the investigation of protein amounts in samples that are difficult to analyze with common biochemical techniques, like mass spectrometry, due to problems in sample purification. Finally, I was able to implement both methods to determine the precise localization and estimate the copy numbers of proteins involved in the synaptic vesicle recycling process in IHC ribbon synapses. Glyoxal fixation improved the preservation of a variety of different targets, and the quality of the subsequent immunostainings. This enabled me to image proteins that have been difficult to visualize in the IHC ribbon synapse in the past. Furthermore, using CosiQuant, I could provide first estimates for the copy number of proteins involved in vesicle recycling at the ribbon synapse of IHCs. Combining these data, I was able to generate a preliminary model of the IHC ribbon synapse, containing information about the spatial organization and the abundancy of 19 synaptic proteins, which might be involved in the synaptic vesicle recycling process. Based on this model, I was able to draw assumptions about the functional importance of the investigated proteins. The protein copy number estimates suggested the proteins that might be rate-limiting in the synaptic vesicle recycling process, while the precise protein localization provided information about where exactly exo- and endocytosis take place. Future studies will provide additional information about synaptic proteins, and will thereby increase the accuracy of the model.