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Causal evidence for retina-dependent and -independent visual motion computations in mouse cortex

Abstract

How neuronal computations in the sensory periphery contribute to computations in the cortex is not well understood. We examined this question in the context of visual-motion processing in the retina and primary visual cortex (V1) of mice. We disrupted retinal direction selectivity, either exclusively along the horizontal axis using FRMD7 mutants or along all directions by ablating starburst amacrine cells, and monitored neuronal activity in layer 2/3 of V1 during stimulation with visual motion. In control mice, we found an over-representation of cortical cells preferring posterior visual motion, the dominant motion direction an animal experiences when it moves forward. In mice with disrupted retinal direction selectivity, the over-representation of posterior-motion-preferring cortical cells disappeared, and their responses at higher stimulus speeds were reduced. This work reveals the existence of two functionally distinct, sensory-periphery-dependent and -independent computations of visual motion in the cortex.

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Figure 1: FRMD7 mutation leads to spiking in all directions in posterior-direction-preferring retinal ON-OFF DS cells.
Figure 2: Starburst-cell ablation leads to spiking in all directions in dorsal-direction-preferring retinal ON-OFF DS cells.
Figure 3: Direction selectivity is largely reduced in starburst-ablated retinas.
Figure 4: Starburst ablation reduces direction selectivity in retinal ON-OFF DS cells in vivo.
Figure 5: Disrupting retinal direction selectivity decreases the proportion of posterior-motion-preferring cortical DS cells in FRMD7tm mice.
Figure 6: Disrupting retinal direction selectivity decreases the proportion of posterior-motion-preferring cortical DS cells in starburst-ablated mice.
Figure 7: Posterior component of cortical DS cell responses is reduced at high stimulus speeds in FRMD7tm mice.

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Acknowledgements

We thank J. Letzkus, M. Hübener, A. Kreile, G. Keller, J. Randall and A. Holtmaat for helping with two-photon imaging and surgery; N. Cesarovic for advice concerning anesthesia; F. Franke and U. Mueller for helping with spike sorting; C. Patino Alvarez for helping with virus production; P. Argast for technical assistance; and S. Oakeley, A. Wertz and A. Attinger for commenting on the manuscript. We acknowledge the following grants: Marie Curie IEF and EMBO LTF to D.H.; Boehringer Ingelheim Fonds PhD fellowship for A.D.; Human Frontier Science Program Postdoctoral Fellowship and Ambizione Fellowship to S.T.; Swiss National Science Foundation, European Research Council, National Centres of Competence in Research Molecular Systems Engineering, Swiss National Science Foundation Sinergia, Swiss-Hungarian, DARPA and European Union 3X3D Imaging grants to B.R. The ETH Zurich group, M.F. and A.H. acknowledge funding through the European Research Council Advanced Grant NeuroCMOS, contract number AdG 267351, and through the Swiss National Science Foundation Sinergia Project CRSII3_141801.

Author information

Authors and Affiliations

Authors

Contributions

D.H. designed, performed and analyzed cortical and LGN experiments; analyzed retinal experiments; performed optokinetic reflex experiments; guided the design and development of software; wrote the real-time response-detection algorithm; performed retinal immunohistochemistry; and wrote the paper. M.F. designed, performed and analyzed retinal experiments. A.D. performed and analyzed retinal experiments on Drd4-GFP and Hb9-GFP mice. S.T. performed cortical experiments and pupillary and optokinetic reflex recordings, and analyzed optokinetic reflex data. Z.R. developed software. G.K. and B. Rozsa developed the 3D two-photon microscope, and G.K., D.H. and B. Rozsa developed corresponding software. S.B.R. performed cortex immunohistochemistry. J.J. developed and made the AAVs. A.H. guided the design and development of the microelectrode arrays. B. Roska designed experiments, analyzed data 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 FRMD7 mutation leads to spiking in all directions in posterior-motion-preferring retinal ON–OFF DS cells.

(a-b) Targeted patch clamp recordings in the retina. Control mice: Drd4-GFP, mutant mice: Drd4-GFP × FRMD7tm. Example responses from GFP-labeled ganglion cells in control (a) and mutant (b) retinas to a stimulus moving in eight directions (black arrows at the bottom). Each block shows the response of a cell to motion in one of eight different directions. Each block has five rows representing five responses of the same cell to repeated stimulus presentations. Vertical lines mark spike occurrences.

Supplementary Figure 2 Ablation of starburst cells in the retina.

ChAT staining labels starburst cells (white dots show cell bodies) in control (top) and in starburst-ablated (bottom) whole-mount mouse retina. The white dots in starburst-ablated retina are fluorescent aggregates and not cell bodies. No cell bodies are present in starburst-ablated retinas.

Supplementary Figure 3 DT injection to the eye selectively ablates starburst cells but leaves other cell populations intact.

The number of retinal cells of a given type or class in control and starburst-ablated retinas. Left, retinal section; middle, top view from confocal projection at the levels marked left to the retinal section by the grey bar; right, quantification. “n” refers to the number of retinas. Starburst cells are GABAergic cells; half of the starburst cells have cell bodies in the ganglion cell layer, which explains the reduction in the number of cell bodies in that layer. Due to the large number of faintly stained GABA cells, this population was not quantified. Note that a brightly labeled subset of GABAergic cells disappeared in the starburst-ablated retina. PL, photoreceptor layer; OPL, outer plexiform layer; INL, inner nuclear layer; GL, ganglion cell layer.

Supplementary Figure 4 Starburst-cell ablation leads to spiking in all directions in dorsal-motion-preferring retinal ON–OFF DS cells.

(a-b) Targeted patch clamp recordings in the retina. Control mice: Hb9-GFP injected with DT, starburst-ablated mice: Hb9-GFP × ChAT-Cre × LSL-DTR injected with DT. Example responses from GFP-labeled ganglion cells in control (a) and starburst-ablated (b) retinas to the stimulus moving in eight directions (black arrows at the bottom). Each block shows the response of a cell to motion in one of eight different directions. Each block has five rows representing five responses of the same cell to repeated stimulus presentations. Vertical lines mark spike occurrences.

Supplementary Figure 5 Effect of starburst-cell ablation on visual reflexes.

(a-b) Pupil reflex test. (a) The pupil of a starburst-ablated mouse is shown in the dark (left) and after a flash of light (right). The extent of the pupil is indicated in white. (b) Quantification of pupil reflex, the relative decrease in pupil diameter after a flash of light, is shown for control and starburst-ablated mice. Black dots: data from individual mice. (c-d) Optokinetic reflex test. (c) Mice were stimulated with gratings moving in a horizontal direction (left). Eye position in response to stimulation before (middle), and seven days after (right) DT injection in the same mouse. Negative deflections indicate eye movements in the anterior (A) direction, while positive deflections indicate eye movements in the posterior (P) direction. (d) Quantification of optokinetic reflex: eye-tracking movements (ETMs) per minute in control and starburst-ablated mice. Black dots: data from individual mice.

Supplementary Figure 6 DTR-expressing ChAT cells in V1 are not affected by DT injection to the eye.

tdTomato-labeled cells in V1 in uninjected (a) and DT-injected (b) ChAT-Cre × LSL-DTR × LSL-tdTomato mice.

Supplementary Figure 7 Disrupting retinal direction selectivity decreases the mean response amplitudes of posterior-motion-preferring cortical DS cells in FRMD7tm and starburst-ablated mice.

(a,d) Polar plots showing the normalized mean of the peak responses (Methods) of cortical DS cells in each of the stimulus directions in FRMD7tm and its control (a) and starburst-ablated and its control (d) mice. Cells with DSI > 0.5 are included in the plot. P denotes posterior, D dorsal, A anterior, V ventral motion direction in the visual field. (b,e) Top, normalized mean of the peak responses in posterior direction of motion in FRMD7tm and its control (b) and starburst-ablated and its control (e) mice. Dark curves show mean values, shaded areas show ±s.e.m around the means. Bottom, the logarithm of p values comparing the conditions using Fisher’s exact test. Values above the magenta line are non-significant. The DSI values shown along the horizontal axis denote the DSI thresholds defining cells as direction selective. (c,f) Top, horizontal versus vertical direction selectivity index computed from the normalized mean peak responses of cortical DS cells along horizontal and vertical directions in control and mutant mice (Methods). Bottom, the logarithm of p values comparing the conditions using Fisher’s exact test. Values above the magenta line are non-significant. The DSI values shown along the horizontal axis denote the DSI thresholds defining cells as direction selective.

Supplementary Figure 8 Disrupting retinal direction selectivity decreases the proportion of posterior-motion-preferring cortical DS cells in FRMD7tm mice when less-reliably responding neurons are included.

Cell inclusion criteria were relaxed to include unreliable cells responding to only one stimulus repetition. (a,d) Polar plot showing the proportion of cortical DS cells preferring each of the stimulus directions in FRMD7tm and its control (a) and starburst-ablated and its control (d) mice. The proportions are normalized to the largest proportion across the two conditions. Cells with DSI > 0.5 are included in the plot. P denotes posterior, D dorsal, A anterior, V ventral motion direction in the visual field. (b,e) Top, proportion of cortical DS cells preferring posterior motion in FRMD7tm and its control (b) and starburst-ablated and its control (e) mice. Dark curves show mean values, shaded areas show ±s.e.m around the means. Bottom, the logarithm of p values comparing the conditions using Fisher’s exact test. Values above the magenta line are non-significant. The DSI values shown along the horizontal axis denote the DSI thresholds defining cells as direction selective. (c,f) Top, horizontal versus vertical direction selectivity index in FRMD7tm and its control (c) and starburst-ablated and its control (f) mice. Bottom, the logarithm of p values comparing the conditions using Fisher’s exact test. Values above the magenta line are non-significant. The DSI values shown along the horizontal axis denote the DSI thresholds defining cells as direction selective.

Supplementary Figure 9 Disrupting retinal direction selectivity decreases the proportion of posterior-motion-preferring cortical DS cells in FRMD7tm mice.

The dependence on the number of stimulus repetitions. The stimulus was presented six times and each row shows response analysis using a subset (two (top row) to six (bottom row)) of stimulus repetitions. (a-o) Control mice: wild type, mutant mice: FRMD7tm. (a,d,g,j,m) Polar plots showing the proportion of cortical DS cells preferring each of the stimulus directions in control and mutant mice. The proportions are normalized to the largest proportion across the two conditions. Cells with DSI > 0.5 are included in the plot. P denotes posterior, D dorsal, A anterior, V ventral motion direction in the visual field. (b,e,h,k,n) Top, proportion of cortical DS cells preferring posterior motion in control and mutant mice. Dark curves show mean values, shaded areas show ±s.e.m around the means. Bottom, the logarithm of p values comparing the conditions using Fisher’s exact test. Values above the magenta line are non-significant. The DSI values shown along the horizontal axis denote the DSI thresholds defining cells as direction selective. (c,f,i,l,o) Top, horizontal versus vertical direction selectivity index in control and mutant mice. Bottom, the logarithm of p values comparing the conditions using Fisher’s exact test. Values above the magenta line are non-significant. The DSI values shown along the horizontal axis denote the DSI thresholds defining cells as direction selective.

Supplementary Figure 10 Response properties of cortical cells in wild-type mice intravenously injected with AAV serotype PHP.B.

(a) Polar plot showing the proportion of cortical DS cells in each of the stimulus directions in control mice. The proportions are normalized to the largest proportion across the two conditions. Cells with DSI > 0.5 are included in the plot. P denotes posterior, D dorsal, A anterior, V ventral motion direction in the visual field. (b) Complementary cumulative distribution of DSI values of recorded cells. Dark curves show mean values, shaded areas show ±s.e.m around the means. (c) Complementary cumulative distribution of OSI values of recorded cells.

Supplementary Figure 11 Intravenous injection of AAV serotype PHP.B yields widespread expression of GCaMP6s in the cortex without nuclear expression.

Sagittal view of V1. (a) Staining with anti-GFP antibody shows cells expressing GCaMP6s. (Reproduced from Figure 7a.) (b) PV-expressing cells stained with anti-PV antibody. (c) Nuclei of cortical cells stained with Hoechst. (d) Merge of (a-c) shows cells coexpressing GCaMP6s and PV.

Supplementary Figure 12 Responding cells are visible in consecutively recorded depths.

(a) Top row: Two-photon image of a cell body in layer 2/3 (left) and its response to motion in eight directions (right). The black part of the grey trace shows the response during image motion. Black arrows below the traces show the stimulus direction. Bottom row: two-photon image and response of the same cell body imaged 10 μm deeper. (b) Top row: Two-photon image of another cell body in layer 2/3 (left) and its response to motion in eight directions (right). Bottom row: two-photon image and response of the same cell body imaged 10 μm deeper.

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Hillier, D., Fiscella, M., Drinnenberg, A. et al. Causal evidence for retina-dependent and -independent visual motion computations in mouse cortex. Nat Neurosci 20, 960–968 (2017). https://doi.org/10.1038/nn.4566

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