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Rods in daylight act as relay cells for cone-driven horizontal cell–mediated surround inhibition

Abstract

Vertebrate vision relies on two types of photoreceptors, rods and cones, which signal increments in light intensity with graded hyperpolarizations. Rods operate in the lower range of light intensities while cones operate at brighter intensities. The receptive fields of both photoreceptors exhibit antagonistic center-surround organization. Here we show that at bright light levels, mouse rods act as relay cells for cone-driven horizontal cell–mediated surround inhibition. In response to large, bright stimuli that activate their surrounds, rods depolarize. Rod depolarization increases with stimulus size, and its action spectrum matches that of cones. Rod responses at high light levels are abolished in mice with nonfunctional cones and when horizontal cells are reversibly inactivated. Rod depolarization is conveyed to the inner retina via postsynaptic circuit elements, namely the rod bipolar cells. Our results show that the retinal circuitry repurposes rods, when they are not directly sensing light, to relay cone-driven surround inhibition.

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Figure 1: Rods respond with depolarization to large spots at high light levels in wholemount retina.
Figure 2: Cone light responses are necessary for rod depolarization.
Figure 3: Blocking photoreceptor input to horizontal cells abolishes rod depolarization.
Figure 4: Reversible inactivation of horizontal cells abolishes depolarizing light responses in rod photoreceptors.
Figure 5: The influence of HEPES on the depolarizing rod response.
Figure 6: Computational modeling of the cone–horizontal cell–rod circuit.
Figure 7: Cone responses at different background intensities in wholemount retina.
Figure 8: Depolarizing rod responses are carried to rod bipolar cells and to the inner retina.

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Acknowledgements

We thank C. Cepko, L. Vandenberghe and S. Rompani for help with high-yield AAV production and P. King, S. Oakeley and members of the Roska laboratory for commenting on the manuscript. We acknowledge the following grants: a Human Frontier Science Program fellowship to S.T.; Boehringer Ingelheim Fonds fellowship to A.D.; Gebert-Ruf Foundation, Swiss National Science Foundation, European Research Council, Swiss-Hungarian National Centres of Competence in Research Molecular Systems Engineering, Sinergia and European Union SEEBETTER, TREATRUSH, OPTONEURO and 3X3D Imaging grants to B.R.; and a Sinergia grant and Centre National de la Recherche Scientifique through UMR 8550 to R.A.S.

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Authors

Contributions

T.S. performed all experiments with rods and cones, recorded from horizontal and rod bipolar cells, did injections, designed experiments, built the two-photon microscope and wrote the paper. S.T. recorded from horizontal and rod bipolar cells. A.D. did AAV injections and quantified the horizontal cell AAV infections. J.J. designed and made the AAV vectors and did AAV injections. Z.R. developed the stimulation and recording software. K.F. built the two-photon microscope and recorded from ganglion cells. M.B. provided the Cnga3−/− mouse. G.A. designed experiments. D.A.C. developed and implemented the quantitative model and performed simulations. J.-A.S. suggested the experiments with the spectrally opponent rod pathway. R.A.d.S. developed the quantitative model, designed experiments and wrote the paper. B.R. designed experiments, implemented the model and wrote the paper.

Corresponding author

Correspondence to Botond Roska.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Light responses of rod photoreceptors in mouse whole mount retina.

a, First column shows the mean and standard error of rod depolarization evoked by a 800 μm spot at 1090 R*/s intensity. In the second and third column rod depolarization to 800 μm spot are compared in two different experimental conditions. Second column: rod depolarization was recorded immediately after getting electrical access to rods. Third column: rod depolarization was recorded at the end of the experimental series shown in Fig. 1a. Significance was tested by Mann-Whitney U test, p = 0.37. b, Normalized response magnitudes in whole mount rods at five background intensities and different spot sizes. c, Rod depolarization at high background light levels increased with increasing positive contrast. d, Rods responded with hyperpolarization to negative contrast, and the hyperpolarization increased with increasing negative contrast.

Supplementary Figure 2 Rod responses in Gnat2cpfl3 mouse.

a, Rod responses at two different background intensities in Gnat2cpfl3 mice. The stimulus was a spot of 800 μm. b, Quantification, responses were normalized to the maximum hyperpolarization evoked by the 0.26 R*/s stimulus.

Supplementary Figure 3 Channelrhodopsin expression in horizontal cells.

We delivered a bi-stable channelrhodopsin (bi-ChR2) to horizontal cells using conditional adeno-associated viruses. A single subretinal injection led to the labeling of horizontal cells across the entire retina.

Supplementary Figure 4 Sequence of light stimulation in the experiments using reversible inactivation of horizontal cells.

First, the retina was stimulated with a test flash, a white spot of 800 µm in diameter shown for 2s. The test flash was not bright enough to activate bi-ChR2 (Fig. 4j). Second, a switch flash was presented. The switch flash was a blue full-field light step that lasted 50 ms. The switch flash was brighter than the test flash and it activated bi-ChR2 (Fig. 4j). Third, a test flash was presented again 10s after the switch flash. After waiting for 5 min we started a new “test flash-switch flash-test flash” cycle. The cycle was then repeated.

Supplementary Figure 5 The influence of picrotoxin and strychnine on the depolarizing rod response.

The effect of the GABA receptor blocker picrotoxin and the glycine receptor blocker strychnine on the depolarizing rod response evoked by a 800 µm diameter spot at 1090 R*/s intensity.

Supplementary Figure 6 The ‘seesaw‘ model at daylight intensities.

Light stimulus leads to cone and, subsequently, horizontal cell hyperpolarization. Horizontal cell hyperpolarization, in turn, depolarizes rods via a sign-inverting synapse.

Supplementary Figure 7 Layout of the upright two-photon microscope.

Supplementary Figure 8 Layout of the optical path.

A two-photon laser source provided a laser beam, which was attenuated by polarization optics and was scanned using mirrors mounted on an upright microscope. The fluorescent signal from labeled cells was split and detected by two photomultipliers. An infrared camera was used to visualize the patch electrode and retinal cells. Infrared light, light for photoreceptor stimulation and light for bi-ChR2 stimulation were provided by a DLP projector. The light provided by the DLP projector was gated by a fast shutter and was modified by neutral density and band pass filters.

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Szikra, T., Trenholm, S., Drinnenberg, A. et al. Rods in daylight act as relay cells for cone-driven horizontal cell–mediated surround inhibition. Nat Neurosci 17, 1728–1735 (2014). https://doi.org/10.1038/nn.3852

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