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Jun Zhang, Alioscka Sousa, Richard D Leapman, Jeffrey Diamond; Electron tomography reveals 3D architecture of the cone ribbon synapse. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2368.
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© ARVO (1962-2015); The Authors (2016-present)
Previous studies suggested that the readily releasable pool (RRP) at ribbon synapses (RSs) in cone consists of 20 (salamander) ~36 (primate) synaptic vesicles (SVs) that can be released rapidly in response to membrane depolarization. To better understand the physical arrangement of the RRP at cone terminals, we used scanning transmission electron tomography (ET) to quantify and visualize the SVs tethered to the ribbon, the subset of those SVs that are docked at the base of the ribbon, and also synaptic ribbon morphology.
ET of cone RSs was performed on routine EM-embedded retinas from P18 Sprague-Dawley rats. Thick sections were collected on Formvar-coated slot grids, counterstained with heavy metals, covered with evaporated carbon, and coated with 10 or 20 nm gold particles. Dual-axis tilt series of selected RSs were acquired using an FEI Tecnai TF30 TEM (at 300-kV beam voltage) from 0.2 µm thick sections with a tilt range of +65° to −65° in 2° angular increments, and from 1.0-1.2 µm thick sections with a tilt range of +55° to −55° in 1.5° angular increments. Images were acquired with pixel sizes of either 0.75 or 1.4 nm. Tilt series were aligned and reconstructed by means of IMOD software, and were then rendered, segmented, and analyzed using Amira software.
Seven entire cone RSs were analyzed. The ribbon at each synapse exhibited plate-like, rectangular morphology, with average reconstructed dimensions of 310±94.1 nm (length; mean±SD) x 236±45.7 nm (height) x 40.4±0.8 nm (width). Each ribbon was tethered to 84±27.2 SVs by thin filaments; 37±2.7% (30±9.3) of those SVs either touched or were tethered to the presynaptic membrane and constituted the RRP. Most SVs in the RRP were at the base of the ribbon, but a few SVs in the middle of the ribbon formed tethers with the presynaptic membrane. SVs had diameters of 38.5±6.5 nm (n=772) and were tethered to the ribbon by several filaments (27.1±5.2 nm in length, n=62) or to the presynaptic membrane by short filaments (11.2±5.1 nm, n=62).
This study imaged, for the first time, the full 3D architecture of mammalian cone RSs, in which regular docked and tethered SVs, as well as plate-like, rectangular-shaped synaptic ribbon were visualized. Quantitative analysis revealed an RRP size that is consistent with previous results and indicates a morphological correlate of the functionally defined RRP.
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