June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Properties of release from rods revealed by visualizing individual synaptic vesicles
Author Affiliations & Notes
  • Minghui Chen
    Department of Ophthalmology & Visual Sciences, University of Nebraska Medical Center, Omaha, NE
    Department of Pharmacology & Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE
  • Matthew Van Hook
    Department of Ophthalmology & Visual Sciences, University of Nebraska Medical Center, Omaha, NE
  • David Zenisek
    Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT
  • Wallace Thoreson
    Department of Ophthalmology & Visual Sciences, University of Nebraska Medical Center, Omaha, NE
    Department of Pharmacology & Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE
  • Footnotes
    Commercial Relationships Minghui Chen, None; Matthew Van Hook, None; David Zenisek, None; Wallace Thoreson, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 1755. doi:
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      Minghui Chen, Matthew Van Hook, David Zenisek, Wallace Thoreson; Properties of release from rods revealed by visualizing individual synaptic vesicles. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1755.

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

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Abstract
 
Purpose
 

Vesicle release from rods is triggered by Ca2+ entry through L-type channels near synaptic ribbons but release may also occur at non-ribbon sites. We visualized fusion of individual synaptic vesicles to characterize sites and kinetics of vesicle release from rods.

 
Methods
 

A small number (1~3%) of vesicles were loaded by briefly incubating salamander retinas with FM1-43 or a dextran-conjugated, pH-sensitive rhodamine, pHrodo in darkness. Synaptic vesicles of isolated rods were imaged using total internal reflection fluorescence microscopy (fig.). Release was stimulated by depolarizing steps applied to voltage-clamped rods or by puffing high KCl or ryanodine.

 
Results
 

Labeled organelles matched the diffraction-limited size of 40-nm microspheres and disappeared rapidly upon stimulation. Disappearance of fluorescent organelles was blocked by inhibiting Ca2+ influx. Kinetics of release measured optically matches that measured from capacitance change. We saw few membrane-associated vesicles unless Ca2+ entry was inhibited. During stimulation, newly appearing vesicles approached the membrane at ~800 nm/s where they paused for ~60 ms before fusion. With fusion, vesicles advanced ~18 nm closer to the membrane. Release occurred mostly near ribbons, but lengthy depolarization stimulated spread of Ca2+ throughout the terminal and caused more release from non-ribbon sites. Activation of Ca2+-induced Ca2+ release (CICR) by ryanodine (10 μM) stimulated Ca2+ increases and vesicle release. Depolarization-evoked Ca2+ spread and ryanodine-evoked effects were inhibited by blocking CICR with dantrolene (10 μM, in bath). Consistent with more release from non-ribbon sites during slow release, damaging the ribbon by fluorophore-assisted laser inactivation of RIBEYE only weakly inhibited exocytotic capacitance jumps evoked by 200-ms depolarizing steps. By contrast, damaging the ribbon strongly inhibited capacitance jumps evoked by 25-ms steps suggesting fast release relies more on ribbons.

 
Conclusions
 

We found that ongoing release at depolarized resting potentials depletes membrane-associated vesicles. Sustained release rates are therefore limited by vesicle delivery rates and membrane dwell times of ~60 ms. The overall rate of sustained release is amplified by release from non-ribbon sites involving CICR from intracellular stores. This amplification may improve detection of changes in release caused by changing illumination.

  
Keywords: 599 microscopy: light/fluorescence/immunohistochemistry • 728 synapse • 689 retina: distal (photoreceptors, horizontal cells, bipolar cells)  
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