Analysis of Feedback Signaling from Horizontal Cells to Photoreceptors in Mice

  • Arlene A. Hirano
  • Xue Liu
  • Nicholas C. Brecha
  • Steven Barnes
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1753)

Abstract

Genetic manipulation of horizontal cells using a Connexin57-iCre mouse (Cx57-iCre) line combined with calcium imaging is proving to be a valuable method to study horizontal cell feedback inhibition onto photoreceptor terminals. While it is accepted that horizontal cells provide lateral inhibitory feedback to photoreceptors, the cellular mechanisms that underlie this feedback inhibition remain only partially elucidated. Feedback inhibition of photoreceptors acts via modulation of their voltage-gated calcium channels at their synaptic terminal. Calcium imaging of photoreceptors in retinal slices, therefore, reflects the impact of inhibitory feedback from horizontal cells. The development of a Cx57-iCre mouse line permits genetic manipulation of horizontal cells. In wild-type mouse retina, depolarization of horizontal cells by kainate provokes a decrease in photoreceptor Ca2+i, whereas hyperpolarization by NBQX elicits an increase in photoreceptor Ca2+i. These responses indicate increased feedback inhibition occurred when horizontal cells are depolarized, and decreased feedback inhibition, when hyperpolarized. This system was used to test the role of GABA release from horizontal cells in feedback inhibition by the selective elimination of VGAT/VIAAT, the inhibitory amino acid transmitter transporter that loads GABA into the synaptic vesicles of horizontal cells. Combined with calcium imaging of photoreceptors in retinal slices, the knockout of specific proteins, e.g., VGAT, provides a robust technique to test the role of GABA in feedback inhibition by horizontal cells.

Key words

Calcium imaging Fluo-4 Horizontal cell Photoreceptor Cre-dependent VGAT knockout 

Notes

Acknowledgments

This work was supported by NIH Grant EY 15573 (NCB), UCLA Oppenheimer Seed Grant (AH, NCB), the Plum Foundation (SB, NCB), a Veterans Administration Career Scientist Award (NCB), Canadian Institutes of Health Research-Nova Scotia Health Research Foundation Regional Partnership Program Grant MOP10968 (SB), and Natural Sciences and Engineering Research Council of Canada Discovery Award (SB).

Supplementary material

Movie 1

Movie of fluo-4-loaded retinal slice as two pulses of 30 mM [K+]o are administered in the bath. The second high [K+] pulse occurs in presence of 50 μM kainate. Note the increase in fluorescence in the photoreceptor cell bodies distal to the outer plexiform layer . (MP4 4371 kb)

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Arlene A. Hirano
    • 1
    • 2
  • Xue Liu
    • 1
    • 3
  • Nicholas C. Brecha
    • 1
    • 2
    • 4
  • Steven Barnes
    • 1
    • 2
    • 5
  1. 1.Department of NeurobiologyDavid Geffen School of Medicine at UCLALos AngelesUSA
  2. 2.Veterans Administration of Greater Los Angeles Health SystemLos AngelesUSA
  3. 3.Biomaterials and Live Cell Imaging InstituteChongqing University of Science and TechnologyChongqingPeople’s Republic of China
  4. 4.Departments of Medicine and OphthalmologyStein Eye Institute, David Geffen School of Medicine at UCLALos AngelesUSA
  5. 5.Departments of Physiology and Biophysics, Ophthalmology and Visual SciencesDalhousie UniversityHalifaxCanada

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