Analysis of Feedback Signaling from Horizontal Cells to Photoreceptors in Mice

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


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 



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)


  1. 1.
    Euler T, Haverkamp S, Schubert T, Baden T (2014) Retinal bipolar cells: elementary building blocks of vision. Nat Rev Neurosci 15(8):507–519CrossRefPubMedGoogle Scholar
  2. 2.
    Shekhar K, Lapan SW, Whitney IE, Tran NM, Macosko EZ, Kowalczyk M, Adiconis X, Levin JZ, Nemesh J, Goldman M, McCarroll SA, Cepko CL, Regev A, Sanes JR (2016) Comprehensive classification of retinal bipolar neurons by single-cell Transcriptomics. Cell 166(5):1308–1323. e1330. CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Wässle H (2004) Parallel processing in the mammalian retina. Nat Rev Neurosci 5(10):747–757. CrossRefPubMedGoogle Scholar
  4. 4.
    Masland RH (2012) The neuronal organization of the retina. Neuron 76(2):266–280. CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Vuong HE, Pérez de Sevilla Müller L, Hardi CN, McMahon DG, Brecha NC (2015) Heterogeneous transgene expression in the retinas of the TH-RFP, TH-Cre, TH-BAC-Cre and DAT-Cre mouse lines. Neuroscience 307:319–337. CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Shimshek DR, Kim J, Hübner MR, Spergel DJ, Buchholz F, Casanova E, Stewart AF, Seeburg PH, Sprengel R (2002) Codon-improved Cre recombinase (iCre) expression in the mouse. Genesis 32(1):19–26CrossRefPubMedGoogle Scholar
  7. 7.
    Hirano AA, Liu X, Boulter J, Grove J, Pérez de Sevilla Müller L, Barnes S, Brecha NC (2016) Targeted deletion of vesicular gaba transporter from retinal horizontal cells eliminates feedback modulation of photoreceptor calcium channels. eNeuro 3(2).
  8. 8.
    Thoreson WB, Mangel SC (2012) Lateral interactions in the outer retina. Prog Retin Eye Res 31(5):407–441. CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Hirasawa H, Kaneko A (2003) pH changes in the invaginating synaptic cleft mediate feedback from horizontal cells to cone photoreceptors by modulating Ca2+ channels. J Gen Physiol 122(6):657–671. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Vessey JP, Stratis AK, Daniels BA, Da Silva N, Jonz MG, Lalonde MR, Baldridge WH, Barnes S (2005) Proton-mediated feedback inhibition of presynaptic calcium channels at the cone photoreceptor synapse. J Neurosci 25(16):4108–4117. CrossRefPubMedGoogle Scholar
  11. 11.
    Cadetti L, Thoreson WB (2006) Feedback effects of horizontal cell membrane potential on cone calcium currents studied with simultaneous recordings. J Neurophysiol 95(3):1992–1995. CrossRefPubMedGoogle Scholar
  12. 12.
    Thoreson WB, Babai N, Bartoletti TM (2008) Feedback from horizontal cells to rod photoreceptors in vertebrate retina. J Neurosci 28(22):5691–5695. CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Babai N, Thoreson WB (2009) Horizontal cell feedback regulates calcium currents and intracellular calcium levels in rod photoreceptors of salamander and mouse retina. J Physiol 587(Pt 10):2353–2364. CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Fahrenfort I, Steijaert M, Sjoerdsma T, Vickers E, Ripps H, van Asselt J, Endeman D, Klooster J, Numan R, ten Eikelder H, von Gersdorff H, Kamermans M (2009) Hemichannel-mediated and pH-based feedback from horizontal cells to cones in the vertebrate retina. PLoS One 4(6):e6090. CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Liu X, Hirano AA, Sun X, Brecha NC, Barnes S (2013) Calcium channels in rat horizontal cells regulate feedback inhibition of photoreceptors through an unconventional GABA- and pH-sensitive mechanism. J Physiol 591(13):3309–3324. CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Kemmler R, Schultz K, Dedek K, Euler T, Schubert T (2014) Differential regulation of cone calcium signals by different horizontal cell feedback mechanisms in the mouse retina. J Neurosci 34(35):11826–11843. CrossRefPubMedGoogle Scholar
  17. 17.
    Schubert T, Weiler R, Feigenspan A (2006) Intracellular calcium is regulated by different pathways in horizontal cells of the mouse retina. J Neurophysiol 96(3):1278–1292. CrossRefPubMedGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Arlene A. Hirano
    • 1
    • 2
    Email author
  • 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

Personalised recommendations