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Gayle J T Pauer, Glenn Lobo, Adrian Au, Stephanie A Hagstrom; Photoreceptor Synaptic Vesicle Cycling in tulp1-/- Mice. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):443.
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© ARVO (1962-2015); The Authors (2016-present)
Tulp1 is a retina-specific protein that localizes to the inner segment (IS), connecting cilium, perikarya and synaptic terminal (ST) of photoreceptor cells. We hypothesize that Tulp1 functions in vesicular trafficking in both the IS and ST. To directly analyze vesicle cycling, we compared tulp1-/- versus wild-type (wt) STs using styryl dye (FM1-43X) photo-conversion followed by electron microscopy (EM).
Eyes were harvested from P16 dark-adapted mice under infrared light. Photoreceptor STs were labeled in the dark by loading 45 uM FM1-43X styryl dye to the intact retina in normal saline containing 1.5 mM Ca2+. Loading was followed by washing in a Ca2+-free solution containing 1 mM EGTA and 1 mM Advasep-7. Retinal slices were assessed for styryl dye uptake and imaged using confocal microscopy. To observe vesicle number and location at the ribbon synapse, retinas were fixed in a 2.5% glutaraldehyde/4% paraformaldehyde solution and incubated in 1 mg/ml diaminobenzidine (DAB). Photo-conversion of DAB to form an electron-dense reaction product was achieved focusing high power 488 nm light onto the tissue with confocal microscopy. Following photo-conversion, the retinal section was prepared and imaged using EM.
In both wt and tulp1-/- retinal sections, we observed strong FM1-43X styryl dye uptake and labeling of photoreceptor inner and outer segments and cell bodies. While wt photoreceptor STs showed strong FM1-43X styryl dye uptake, tulp1-/- photoreceptor STs were significantly reduced of styryl dye staining. EM images of wt retinal sections revealed photoreceptor STs to be packed with small electron dense vesicles that were tightly tethered to the ribbon. In contrast, EM images of tulp1-/- retinal sections showed much fewer electron dense vesicles at the STs, most of which did not tether to the synaptic ribbons.
Our data suggests that tulp1 is required for vesicle cycling in the STs of photoreceptor cells. In addition, in the absence of tulp1, vesicle docking on the ribbon synapse does not appear to occur correctly, suggesting a possible explanation for poor synaptic transmission observed in tulp1-/- mice.
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