July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Chemogenetic manipulation of feedback inhibition from horizontal cells to photoreceptors in transgenic zebrafish expressing alien neurotransmitter receptors
Author Affiliations & Notes
  • Billie Beckwith-Cohen
    Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, United States
  • Lars Holzhausen
    Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, United States
  • Richard H Kramer
    Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, United States
  • Footnotes
    Commercial Relationships   Billie Beckwith-Cohen, None; Lars Holzhausen, None; Richard Kramer, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 603. doi:https://doi.org/
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      Billie Beckwith-Cohen, Lars Holzhausen, Richard H Kramer; Chemogenetic manipulation of feedback inhibition from horizontal cells to photoreceptors in transgenic zebrafish expressing alien neurotransmitter receptors. Invest. Ophthalmol. Vis. Sci. 2018;59(9):603. doi: https://doi.org/.

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

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Abstract

Purpose : Despite 70 years of work on retinal neural circuits, questions remain about the role of horizontal cells (HCs) in information processing. Here we explore how selective chemogenetic manipulation of horizontal cell voltage affects retinal signals using transgenic zebrafish models.

Methods : We genetically engineered lines of zebrafish with alien receptors that affect the membrane potential when activated by their cognate agonists. For depolarizing HCs, we used a FMRFamide-gated Na+ channel (FaNaC). For hyperpolarizing HCs, we use a “Designer Receptor Exclusively Activated by a Designer Drug” (a DREADD, named PSAM) derived from a modified, Cl- conducting glycine receptor. Selective HC expression was achieved with a cell-type specific promoter (connexin-55.5). FaNaC and PSAM fish lines were cross-bred to introduce the genetically encoded calcium indicator GCaMP6f, expressed in RGCs with a neuronal promoter (HuC). To monitor the effects of manipulating HC voltage on photoreceptor synaptic transmission, we imaged the release of the synaptic vesicle fluorescent dye FM1-43, and recorded electroretinograms (ERGs) from eyes of 4-6 dpf larva, before and after the application of FMRFamide or the PSAM agonist (PSEM-89s). To monitor effects of these manipulations on RGCs, we used 2-photon calcium imaging of retinal whole-mounts.

Results : Transgenic FaNaC and PSAM-GlyR larvae exhibit normal ERGs in the absence of an agonist. The application of 30μM FMRFamide caused a decrease in peak B-wave amplitude in FaNaC fish (-35%), but not in wild type. The application of 30μM PSEM-89s also caused a decrease in peak B-wave amplitude of PSAM-GlyR fish (-15%), but not in wild type. The rate of FM1-43 destaining showed a 2.5 fold acceleration in PSAM-GlyR fish and 1.5 deceleration in FaNaC fish after application of the cognate agonist. Calcium imaging revealed that HC depolarization with FMRFamide or hyperpolarization with PSEM-89s interfered with the normal center surround response of RGCs.

Conclusions : Selective chemogenetic manipulation of the membrane potential of HCs provides a new opportunity for understanding their role in retinal information processing (e.g. long-term light adaptation, contrast sensitivity, and color opponency), both in normal vision and in pathological state.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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