Analysis of Feedback Signaling from Horizontal Cells to Photoreceptors in Mice
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 wordsCalcium 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).
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)
- 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. https://doi.org/10.1016/j.cell.2016.07.054 CrossRefPubMedPubMedCentralGoogle Scholar
- 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. https://doi.org/10.1016/j.neuroscience.2015.08.060 CrossRefPubMedPubMedCentralGoogle Scholar
- 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). https://doi.org/10.1523/ENEURO.0148-1515.2016
- 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. https://doi.org/10.1523/JNEUROSCI.5253-04.2005 CrossRefPubMedGoogle Scholar
- 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. https://doi.org/10.1371/journal.pone.0006090 CrossRefPubMedPubMedCentralGoogle Scholar
- 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. https://doi.org/10.1113/jphysiol.2012.248179 CrossRefPubMedPubMedCentralGoogle Scholar