June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Optogenetic detection of spatial inhomogeneity of protons in the synaptic cleft during horizontal cell to cone feedback underlying lateral inhibition
Author Affiliations & Notes
  • Billie Beckwith-Cohen
    Vision Science, University of California, Berkeley, Berkeley, California, United States
  • Lars Holzhausen
    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, United States
  • Richard Kramer
    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, United States
    Vision Science, University of California, Berkeley, Berkeley, California, United States
  • Footnotes
    Commercial Relationships   Billie Beckwith-Cohen, None; Lars Holzhausen, None; Richard Kramer, None
  • Footnotes
    Support  NIH Grant EY024334
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 2977. doi:
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    • Get Citation

      Billie Beckwith-Cohen, Lars Holzhausen, Richard Kramer; Optogenetic detection of spatial inhomogeneity of protons in the synaptic cleft during horizontal cell to cone feedback underlying lateral inhibition. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2977.

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

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Abstract

Purpose : Recent work utilizing an optogenetic pH sensor has implicated protons as mediators of lateral inhibition in the zebrafish retina. We explore the spatial distribution of protons in the invaginating cone synapse with three 3 distinct optogenetic pH-sensors displayed at different subcellular locations within the synaptic cleft.

Methods : We genetically engineered 3 lines of zebrafish that express pH-sensitive GFP probes (pHluorins) fused onto different synaptic proteins in either cones or horizontal cells (HCs). We used the cone transducin promoter for cell-type specific expression of SynaptopHluorin (pHluorin fused onto the synaptic vesicle protein synaptobrevin) or CalipHluorin (pHluorin fused onto an accessory subunit of the L-type Ca channel). In HCs, we used the connexin-55 promoter to express AMPApHluorin (pHluorin fused to the GluR2 subunit of the AMPA receptor). Light-elicited changes in synaptic cleft pH were measured with 2-photon imaging of retinal whole-mounts.

Results : In retinas expressing each pH probes, fluorescence intensity was up to 5% brighter immediately after a delivered light flash. The signal was blocked by HEPES, consistent with a change in free proton concentration (increase in pH) triggered by light. Fluorescence change increased with spot diameter and was seen with an annulus, observations consistent with HC feedback. As expected, the signal was eliminated with GYKI, an antagonist of ionotropic glutamate receptors found on HCs but not on photoreceptors. Full-field illumination for 500 msec caused a 5% increase in fluorescence with CalipHluorin, 1.8% increase with SynaptopHluorin, and 1% increase with AMPApHluorin (p<0.05). The pHluorin in each probe was identical, but exhibited a different expression pattern in the invaginating cleft. CalipHluorin and was highly localized to the center of the cleft, SynaptopHluorin was diffusely distributed, and AMPApHluorin was expressed predominantly in the cleft perimeter.

Conclusions : We measured light-elicited pH changes underlying lateral inhibition mediated by HC feedback. The proton gradient observed in the synaptic cleft raises a possibility that the source from which protons (or alternatively, a proton buffer) emanate lies central within the invaginating cleft and away from HC terminal perimeter.

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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