June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Assessing Contributions of Kiss-and-run to Synaptic Exocytosis and Endocytosis from Photoreceptors with Optical Measurements of Fusion Pore Size
Author Affiliations & Notes
  • Xiangyi Wen
    University of Nebraska Medical Center, Omaha, Nebraska, United States
  • Grant Saltzgaber
    University of Nebraska Medical Center, Omaha, Nebraska, United States
  • Wallace B. Thoreson
    University of Nebraska Medical Center, Omaha, Nebraska, United States
  • Footnotes
    Commercial Relationships   Xiangyi Wen, None; Grant Saltzgaber, None; Wallace Thoreson, None
  • Footnotes
    Support  NIH Grant EY10542, Research to Prevent Blindness, China Scholarship Council
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 5606. doi:
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      Xiangyi Wen, Grant Saltzgaber, Wallace B. Thoreson; Assessing Contributions of Kiss-and-run to Synaptic Exocytosis and Endocytosis from Photoreceptors with Optical Measurements of Fusion Pore Size. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5606.

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

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Abstract

Purpose : Photoreceptors release glutamate continuously in darkness. Neurotransmitters can be released by kiss-and-run or full-collapse fusion of synaptic vesicles. It is unclear how much kiss-and-run contributes to synaptic release from various neurons including photoreceptors. We therefore assessed contributions of kiss-and-run vs. full-collapse to exocytosis and endocytosis in salamander photoreceptors using optical methods.

Methods : We used different size dyes (SR101, 1 nm; 3-kDa dextran-conjugated Texas Red or Alexa Fluor 488, 2.3 nm; 10-kDa Texas Red, Alexa Fluor 488, or pHrodo, 4.6 nm; 70-kDa Texas Red, 12 nm) to measure fusion pore diameter and calculate proportions of kiss-and-run. To measure endocytosis, we incubated rods and cones with various dyes and then measured either single vesicle loading by total internal reflection fluorescence microscopy (TIRFM) or whole terminal fluorescence. To assess exocytosis, we imaged individual vesicles using TIRFM and then distinguished vesicle release events by their rapid disappearance.

Results : After 10 min incubation, the concentration of small dyes (SR101; 3-kDa Texas Red) loaded into rod and cone terminals was 8-12 times higher than large dyes (10-kDa Texas Red, 10-kDa pHrodo, 70-kDa Texas Red) suggesting the existence of kiss-and-run events with fusion pores between 2.3-4.6 nm. Using TIRFM, we found that when rods were co-incubated with Texas Red or Alexa Flour 488 conjugated to 3-kDa or 10-kDa dextran, significantly more vesicles loaded with small dyes than large dyes (P<0.05). When vesicles were loaded with SR101, 3-kDa Texas Red, or pHrodo, the population of release events identified by TIRFM constituted ~20-22% of all measured events. For 10- and 70-kDa Texas Red, this population was only 6-10% of the total. While pHrodo itself cannot permeate a small pore, its fluorescence can be rapidly quenched by proton extrusion during kiss-and-run.

Conclusions : Measurements of exocytosis and endocytosis showed the presence of kiss-and-run with a fusion pore diameter of 2.3-4.6 nm in photoreceptors. Assuming that large dyes can be released only by full-collapse whereas small dyes can be released by both modes, kiss-and-run accounts for >50% of synaptic release from photoreceptors. Thus, kiss-and-run fusion may facilitate continuous release at photoreceptor ribbon synapses.

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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