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H. Regus-Leidig, J. Atorf, A. Feigenspan, S. Boye, V. Chiodo, G. M. Seigel, W. W. Hauswirth, J. Kremers, J. H. Brandstätter; The Photoreceptor Ribbon Synapse in the Absence of Piccolo: An RNAi Approach. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4120.
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© ARVO (1962-2015); The Authors (2016-present)
The mammalian photoreceptor ribbon complex is specialized for the tonic release of neurotransmitter. It separates into two spatially and functionally distinct molecular compartments: the ribbon compartment and the arciform density/plasma membrane compartment. The huge cytomatrix protein Piccolo is located at the ribbon compartment, where it colocalizes and interacts with RIBEYE, the major constituent of the ribbon. As slowly the role of Piccolo is being unravelled at conventional chemical synapses, nothing is known about its function at photoreceptor ribbon synapses. In this study we use RNA interference to specifically knock down Piccolo in photoreceptors already during synaptogenesis.
Targeted delivery of Piccolo specific shRNA is achieved via subretinal injection of shRNA carrying AAV5 in postnatal day 5 old C57/Bl6 mice. For detection of transduced photoreceptors, hGFP is expressed under the control of an opsin promoter.
Knockdown efficiency of Piccolo mRNA was verified in HEK293 cells, R28 cells and isolated transduced photoreceptors. The transduction rate of individual retinae varies between 10% and 100% depending on the distance to the injection site. So far, immunocytochemical analysis indicates that lack of Piccolo results in a drastic reduction in ribbon size, while expression and distribution of other presynaptic molecules, e.g. SNARE and synaptic vesicle proteins, remain unchanged. This indicates a scaffolding role of Piccolo at the photoreceptor ribbon.
As an alternative to the generation of knockout animals, our tissue specific knockdown approach is a very promising tool to study the contribution of presynaptic proteins to photoreceptor ribbon structure and function. Apart from immunocytochemical examinations, in the future our analyses will include electrophysiological and multifocal ERG recordings, as well as immunoelectron microscopy.
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