March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
Three-dimensional Architecture Of The Rod Bipolar Cell Ribbon Synapse
Author Affiliations & Notes
  • Jun Zhang
    Synaptic Physiology Section, NINDS/NIH, Bethesda, Maryland
  • Alioscka Sousa
    Laboratory of Cellular Imaging and Macromolecular Biophysics, NIBIB/NIH, Bethesda, Maryland
  • Richard Leapman
    Laboratory of Cellular Imaging and Macromolecular Biophysics, NIBIB/NIH, Bethesda, Maryland
  • Jeffrey Diamond
    Synaptic Physiology Section, NINDS/NIH, Bethesda, Maryland
  • Footnotes
    Commercial Relationships  Jun Zhang, None; Alioscka Sousa, None; Richard Leapman, None; Jeffrey Diamond, None
  • Footnotes
    Support  NINDS intramural research program
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 4318. doi:
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      Jun Zhang, Alioscka Sousa, Richard Leapman, Jeffrey Diamond; Three-dimensional Architecture Of The Rod Bipolar Cell Ribbon Synapse. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4318.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : Physiological studies suggested that the readily releasable pool (RRP) at ribbon synapses in rod bipolar cells (RBCs) comprises a small population of synaptic vesicles (7 on average) that can be released rapidly in response to membrane depolarization. To better understand the physical arrangement of the RRP at RBC terminals, we used electron tomography (ET) to quantify and visualize the vesicles docked at the base of the ribbon and vesicles tethered to the ribbon.

Methods: : ET of RBC ribbon synapses 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 as fiducial markers. Dual-axis tilt series of selected ribbon synapses 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° and 2° angular increments, and from 1.0-1.2 µm thick sections with a tilt range of +55° to -55° and 1.5° angular increments. Images were acquired with pixel sizes of either 0.75 or 1.4 nm referred to the specimen. Tilt series were aligned and reconstructed by means of IMOD software, and were then rendered, segmented, and analyzed using Amira software.

Results: : Six entire RBC ribbon synapses were analyzed. The ribbon at each synapse exhibited typical plate-like, rectangular morphology, with an average reconstructed volume of 200±28 nm (length; mean±SD) x 150±34 nm (height) x 41±1 nm (width). Ribbons were connected to 38.2±10.0 synaptic vesicles by thin filaments; 25.1±1.1% (9.5±2.4) of those vesicles either touched or were tethered to the presynaptic membrane and constituted the RRP. Most vesicles in the RRP were at the base of the ribbon, but a few vesicles in the middle of the ribbon formed tethers with the presynaptic membrane. Vesicle diameter was 37±6 nm (n=192), although some larger vesicles (60 ~ nm) were observed at the base or in the middle of the ribbon.

Conclusions: : This study determined, for the first time, a full three-dimensional architecture of mammalian RBC ribbon synapses, in which regular docked and tethered vesicles, as well as larger, perhaps pre-fused, vesicles were visualized. Quantitative analysis revealed an RRP size that is consistent with previous physiological results and indicates a morphological correlate of the functionally defined RRP.

Keywords: synapse • bipolar cells • microscopy: electron microscopy 
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