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Wallace B. Thoreson, Matthew J Van Hook, Xiangyi Wen, Karlene M Cork; Endocytosis promotes release site restoration at rod ribbon synapses. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1030.
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© ARVO (1962-2015); The Authors (2016-present)
Synaptic endocytosis is needed to regenerate synaptic vesicles, but may also be important for removing previously used vesicle proteins to restore release site function. We hypothesized that this may be particularly important at synaptic ribbons where vesicles are released continually at closely neighboring sites. To test this, we inhibited endocytosis at rod ribbon synapses.
Post-synaptic currents (PSCs) were evoked in horizontal cells (HCs) by depolarizing simultaneously voltage-clamped rods in salamander retinal slices. Synaptic vesicles were loaded into isolated rods with dextran-conjugated pHrodo and imaged by total internal reflectance fluorescence microscopy (TIRFM). Movements of individual Ca2+ channels were tracked in rods by tagging channels with quantum dots (QDs) conjugated to Ca2+ channel α2δ4 antibodies.
Inhibiting endocytosis with the dynamin inhibitor Dynasore (80 μM) quickly reduced fast, ribbon-mediated components of HC PSCs (test/control: 0.67±0.06, N=10) but not slower non-ribbon components (1.0±0.13) or rod ICa (1.0±0.12). Non-ribbon PSCs and spontaneous mEPSC frequency (but not amplitude) declined after 4 min of Dynasore treatment, perhaps due to vesicle depletion. Inhibiting dynamin with Dynasore (80 μM) or Dyngo (30 μM) introduced through rod patch pipettes slowed recovery of HC PSCs from paired pulse depression from τ=600 ms to 3 s. Bath-applied Dynasore significantly reduced HC currents evoked by 2 Hz sinusoidal 580 nm light (P=0.04). Rod light responses were not altered. Using TIRFM to image individual synaptic vesicles during stimulation with 50 mM K+, Dynasore significantly increased frequency of vesicles docked at the plasma membrane sevenfold (P<0.0001) and reduced fusion events fourfold (P=0.017). During 20 mM K+ application, Dynasore significantly expanded lateral membrane movements of QD-tagged Ca2+ channels (P<0.0001).
Rapid inhibition of fast but not slow PSCs by Dynasore may reflect a greater need for release site clearance at ribbons vs. more dispersed non-ribbon release sites. Paired pulse and light response experiments also showed a role for endocytosis in restoring release sites. TIRFM studies revealed that dynamin inhibition impairs fusion but not docking of newly arrived vesicles. By increasing the distance from channels to release sites, expanding Ca2+ channel confinement domains by dynamin inhibition can reduce release efficiency.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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