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Ted Warren, Claudiu Supuran, Wallace B Thoreson; Sources of protons involved in horizontal cell to cone feedback.. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2610.
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© ARVO (1962-2015); The Authors (2016-present)
Feedback from horizontal cells (HCs) to cones contributes to generation of center-surround receptive fields and color opponency in the retina. HC feedback has been shown to involve protons that influence both voltage-dependence and conductance of L-type Ca2+ currents (ICa) in cones that in turn regulate glutamate release. We analyzed mechanisms and sources of extracellular protons in HC feedback.
We studied HC feedback in tiger salamander retina using two experimental protocols: 1) measured feedback currents in cones evoked by surround illumination in flatmount retina and 2) used paired recordings in retinal slices to measure changes in cone ICa caused by changes in HC holding potential.
We tested three sources for synaptic cleft protons in feedback: 1) We blocked vesicular ATPases with bafilomycin to inhibit proton loading into synaptic vesicles of cones and HCs. Overnight incubation in bafilomycin blocked post-synaptic currents in HCs as expected from vesicular ATPase block, but did not reduce HC feedback strength assessed by protocol 2. 2) We blocked carbonic anhydrase (CA) in the synaptic cleft using membrane-permeant and impermeant CA inhibitors, acetazolamide and FC5-207a. These compounds altered ICa voltage-dependence as expected for CA inhibition but did not weaken feedback assessed by protocol 1. 3) Inhibiting proton extrusion via Na+/H+ exchange by replacing extracellular Na+ with choline rapidly eliminated feedback. Reducing cytoplasmic protons in HCs, but not in cones, with pH 9 pipette solution also eliminated feedback, implicating HCs as the proton source. Acidifying extracellular pH (from 7.8 to 7.4 to 7.1) to increase synaptic cleft protons enhanced feedback assessed by protocol 2, consistent with proton influx into HCs during light-evoked hyperpolarization. Stepping HCs from -30 to -60 mV to simulate light-evoked hyperpolarization evoked inward feedback currents in cones with fast time constants averaging 19+5.9 ms, slower than expected for an ephaptic mechanism.
Our results suggest that Na+/H+ exchange in HCs provide a tonic source of protons to the cone synaptic cleft in darkness. When hyperpolarized by light, HCs may also act as a proton sink (e.g., by influx of protons through ion channels). The relatively slow kinetics of feedback currents in cones stimulated by voltage steps applied to HCs suggests minimal contributions from ephaptic mechanisms.
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