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L. C. Holzhausen, P. Theer, P. B. Detwiler, J. B. Hurley, S. E. Brockerhoff; Imaging of Light-Dependent Vesicle Release in the Cone Photoreceptor Ribbon Synapse. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1269. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Photoreceptor ribbon synapses release glutamate to transmit light responses to secondary neurons. While extensive studies document signal transduction in photoreceptor outer segments, less is known about the formation of cone synapses and how the light signal is transmitted by photoreceptor ribbon synapses to secondary neurons. To study cone photoreceptor ribbon synapses, we developed a transgenic zebrafish line that expresses a pH-sensitive synaptic vesicle marker (synaptopHluorin) specifically in cone photoreceptors. Our goal is to use this zebrafish line to characterize exocytosis and endocytosis at the cone photoreceptor ribbon synapse.
We generated transgenic zebrafish that express synaptopHluorin (spH) under control of the cone transducin promoter to direct expression of spH specifically to cone photoreceptors. spH is a fluorescent indicator of endo- and exocytosis. It consists of a pH-sensitive GFP fused to the luminal C-terminus of the vesicle protein VAMP-2. Fluorescence of spH is quenched in the low pH vesicular environment and becomes dequenched after exocytosis. Therefore, changes in fluorescence in cone synapses will provide a read-out for ongoing endo- and exocytosis. spH fluorescence is excited using 2-photon absorption of long wavelength light (>890nm) to minimize direct photoreceptor activation during the fluorescent measurement. For the analysis, a flattened retina from a transgenic zebrafish is prepared, and we use a LED light stimulus at 590 nm at various light intensities and durations to evoke changes in spH fluorescence, which provides a read out of endo- and exocytosis.
We have made a stable line of transgenic zebrafish that expresses spH specifically in cone photoreceptors. spH fluorescence is present mostly in cone synaptic terminals, but there also is some fluorescence in the cone cell body. Since some VAMP-2 ‘escapes’ the synaptic region, this result could be expected. We have begun to use spH to characterize the light dependence and kinetics of vesicle release by cones. As expected, bright light (6x106 photons/µm2/s) leads to a transient decrease in spH fluorescence due to change in the relative rate of endo- and exocytosis during light exposure. We can detect light-stimulated fluorescent decreases that occur within 128 ms.
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