Experimental animals

Wild-type control mice (C57BL/6J) were obtained from Janvier Labs. Frmd7tm mice are homozygous female or hemizygous male Frmd7tm1b(KOMP)Wtsi mice, which were obtained as Frmd7tm1a(KOMP)Wtsi from the Knockout Mouse Project (KOMP) Repository25,27: Exon 4 and neo cassette flanked by loxP sequences were removed by crossing with female Cre-deleter Edil3Tg(Sox2−cre)1Amc/J mice (Jackson laboratory stock 4783) as confirmed by PCR of genome DNA, and maintained in C57BL/6J background. ChAT-Cre (strain: Chattm2(cre)Lowl/MwarJ, Jackson laboratory stock: 028861)53 and LSL-DTR (strain: Gt(ROSA)26Sortm1(HBEGF)Awai/J, Jackson laboratory stock: 007900)54 were purchased from Jackson laboratory and maintained in C57BL/6J background. Experiments were performed on 34 male and female wild-type control mice, 33 male and female Frmd7tm mice, and 10 female ChAT-Cre × LSL-DTR mice. All mice were between two and four months old. Mice were group-housed and maintained in a 12-h/12-h light/dark cycle with ad libitum access to food and water. Experiments were performed according to standard ethical guidelines and were approved by the Danish National Animal Experiment Committee.

Head-plate and cranial window implantation

Mice were anaesthetized with an intraperitoneal injection of a fentanyl (0.05 mg/kg body weight; Hameln), midazolam (5.0 mg/kg body weight; Hameln) and medetomidine (0.5 mg/kg body weight; Domitor, Orion) mixture dissolved in saline. The depth of anesthesia was monitored by the pinch withdrawal reflex throughout the surgery. Core body temperature was monitored using a rectal probe and temperature maintained at 37−38 °C using a feedback-controlled heating pad (ATC2000, World Precision Instruments). Eyes were protected from dehydration during the surgery with eye ointment (Oculotect Augengel). The scalp overlaying the left visual cortex was removed, and a custom head-fixing imaging head-plate with a circular 8 mm diameter opening was mounted on the skull using cyanoacrylate-based glue (Super Glue Precision, Loctite) and dental cement (Jet Denture Repair Powder) to allow for subsequent head fixation during imaging. The center of the head-plate was positioned above V1, 2.5 mm lateral and 1 mm anterior of lambda55. To gain optical access to the cortex, a 5 mm diameter craniotomy was performed. After removing the skull flap, the cortical surface was kept moist with a cortex buffer containing 125 mM NaCl, 5 mM KCl, 10 mM glucose, 10 mM HEPES, 2 mM MgSO 4 , and 2 mM CaCl 2 . The dura was left intact (except in two animals in which the dura spontaneously detached with the skull flap) and any occasional bleedings were immediately stopped with Gelfoam (Pfizer). A 5 mm glass coverslip sterilized in ethanol (0.15 mm thickness, Warner Instruments) was placed onto the brain to gently compress the underlying cortex and dampen biological motion during subsequent imaging56. The cranial window was hermetically sealed using a cyanoacrylate-based glue (Super Glue Precision, Loctite) mixed with black dental cement (Jet Denture Repair Powder mixed with iron oxide powdered pigment) to prevent the entry of stray light from the screen through the skull and/or cement during imaging56. Mice were returned to their home cage after anesthesia was reversed with an intraperitoneal injection of a flumazenil (0.5 mg/kg body weight; Hameln) and atipamezole (2.5 mg/kg body weight; Antisedan, Orion Pharma) mixture dissolved in saline, and after recovering on a heating pad for one hour.

Intrinsic imaging

For ISOI mice were anesthetized with isoflurane (2−3% induction) and head-fixed in a custom holder. Chlorprothexine was administered intraperitoneally (2.5 mg/kg body weight; Sigma) as a sedative12, and isoflurane reduced to 0.5−1% and kept constant during visual stimulation. Core body temperature was maintained at 37−38 °C using a feedback-controlled heating pad (ATC2000, World Precision Instruments). The stimulated contralateral eye was kept lubricated by hourly application of a thin layer of silicone oil (OFNA Racing, 10,000 molecular weight). The experimental setup employed for ISOI was adapted from a similar system57 and made publicly available (https://snlc.github.io/ISI/). A 2 × air-objective (Olympus, 0.08 NA, 4 mm field of view) was mounted on our Scientifica VivoScope, which was equipped with a CMOS camera (HD1-D-D1312-160-CL-12, PhotonFocus), a large-well-depth camera that offers high signal-to-noise measurements in bright light conditions. The camera was connected to a Matrox Solios (eCL/XCL-B) frame-grabber via Camera Link. The acquisition code for the Matrox board was written in Matlab using the Image Acquisition Toolbox. From the pial surface, the microscope was defocused down 400−600 µm, where intrinsic signals were excited using a red LED (KL1600, Schott) delivered via light guides through a 610 nm long-pass filter (Chroma). Reflected light was captured through a 700 ± 50 nm (mean ± SEM) band-pass filter (Chroma) positioned right in front of the camera at a rate of 6 frames per second (512 × 512 pixels). At 700 nm there is a large change in the absorption coefficient between oxyhemoglobin and deoxyhemoglobin, contributing to the intrinsic signal measured in these experiments12. The 47.65 × 26.87 cm (width × height) screen was angled 30° from the mouse’s midline and positioned so that the perpendicular bisector was 10 cm from the bottom of the screen, centered on the screen left to right (23.8 cm on each side), and 10 cm from the eye57. This resulted in a visual-field coverage from −41.98° to 60.77° (total 102.75°) in elevation and from −67.23° to 67.23° (total 134.46°) in azimuth. Thus, the stimulus covered almost the entire known visual hemi-field of the mouse, which is estimated to be at most 110° vertically and 140° horizontally. For the ISOI experiments presented in Fig. 1, each mouse was imaged on three separate days, and the data averaged to reduce the chance of day-to-day variations confounding group-level results.

Visual stimuli for ISOI

Retinotopic maps were generated by sweeping a spherically corrected (Matlab code provided by Spencer Smith: https://labrigger.com/blog/2012/03/06/mouse-visual-stim/) full-field bar across the screen in both azimuth and elevation directions57. The bar contained a flickering black-and-white checkerboard pattern on a black background7,57. The width of the bar was 12.5° and the checkerboard square size was 25°. Each square alternated between black and white at 4 Hz. In each trial, the bar was drifted 10 times in each of the four cardinal directions (0°, 90°, 180°, and 270°), moving at 8−9°/s. Usually, two to three trials were sufficient to achieve well-defined retinotopic maps. For measuring and characterizing the evoked areal activity in V1 and HVAs we presented 100% contrast black and white sinusoidal gratings drifting in each of the four cardinal directions. We presented gratings with four different TFs: 0.3, 0.75, 1.2, and 1.8 Hz (0.03 cycles/°). Each stimulus was stationary for 10 s and in motion for 10 s, comprising a stimulus period of 20 s, which was repeated 5 times in each direction. All stimuli used for ISOI were produced and presented using Matlab and the Psychophysics Toolbox58.

Image analysis for ISOI

To generate functional visual cortex maps from the raw image data, we took the response time course for each pixel and computed the phase and magnitude of the Fourier transform at the stimulus frequencies (0.067 and 0.088 Hz, for azimuth and vertical, respectively)59. The bar was drifted in opposite directions in order to subtract the delay in the intrinsic signal relative to neuronal activity59. The resulting phase maps were then converted into retinotopic coordinates (visual degrees) from the known geometry of our setup to retrieve absolute retinotopy. We used automated, publicly available code to identify visual area borders based on their visual-field sign maps57 (see also description below), and superimposed those borders with the anatomical blood-vessel images to accurately localize V1 and individual HVAs.

To evaluate and quantify the spatial properties of visual areas, we first computed and identified the borders of each visual area, using a dissociation algorithm and the Image Processing Toolbox in Matlab. The identification process constituted three conventional edge-detection steps. (1) Thresholding of the obtained visual-field sign map. For this we used a definition of \(\bar I + {\mathrm{SD}}\left( I \right)\) where I equals the intensity of a pixel and \(\bar I\) denotes the mean pixel intensity of the visual-field sign map. The thresholded image was smoothed using a median filter (filter size, 3 × 3 neighborhoods). (2) Isolation and segmentation of pixels. For this step we used the 8-neighbors criterion. If there were >4 non-zero pixels among the eight neighbors of one pixel, the pixel was retained and any gaps between pixels within the eight neighbors were filled with a non-zero value. If this criterion was not met, the pixel value was set to 0. The value of all non-zero pixels was set to 1. (3) Edge detection. The isolated and segmented pixels were next binarized, and each edge was computed based on a Sobel method using the edge function in Matlab. After identifying visual area borders, we computed the size of the area (first in pixels and then converted to mm2) and defined the center position of each area as a centroid. From this, we calculated two-dimensional coordinates of each HVA as coordinates relative to the V1 centroid. For ISOI experiments in which sinusoidal gratings were presented, the raw response signal was first determined as the peak power of the stimulus-evoked signal by employing Fast-Fourier transform analyses of each pixel column at the frequencies of the visual stimuli; 0.05−0.1Hz12. To quantify the response for each visual cortical area, the raw response signal was first normalized to the raw response signal from before the visual stimulation (averaged over a 10 s period). Next, regions of interest (ROIs) within the visual cortical areas were defined based on the visual area border map. The response strength of each area was determined as the maximum value within each ROI. For group-level quantifications, the response strength for a given area was averaged across the three experimental days before pooling data from all mice.

Local viral labeling

For local viral injections in the visual cortex or dLGN, mice were first anesthetized with an intraperitoneal injection of a fentanyl (0.05 mg/kg body weight; Hameln), midazolam (5.0 mg/kg body weight; Hameln) and medetomidine (0.5 mg/kg body weight; Domitor, Orion) mixture dissolved in saline. For injections yielding GCaMP6 expression in areas V1, RL and PM, three 0.4 mm diameter craniotomies were performed over the left visual cortex and 100 nl AAV2/1-Syn-GCaMP6f-WPRE (2.13 × 1013 vg/ml, Penn Vector Core #AV-1-PV2822) slowly injected at depths of 100−500 µm using a borosilicate glass micropipette (30 µm tip diameter) and a pressure injection system (Picospritzer III, Parker). For labeling geniculo-cortical axons projecting from the dLGN, 20−40 nl AAV2/1-Syn-GCaMP6f-WPRE (2.13 × 1013 vg/ml, Penn Vector Core #AV-1-PV2822) was slowly injected into the left dLGN using stereotaxic coordinates: 2.1 mm posterior of the Bregma; 2.2 mm lateral of the midline; 2.3 mm below the pial surface23. To prevent backflow during withdrawal, the micropipette was kept in the brain for a minimum of 5 min before it was slowly retracted. The skin was afterwards sutured shut. Mice were returned to their home cage after the anesthesia was reversed with an intraperitoneal injection of a flumazenil (0.5 mg/kg body weight; Hameln) and atipamezole (2.5 mg/kg body weight; Antisedan, Orion Pharma) mixture dissolved in saline, and after recovering on a heating pad for 1 h. Although the injection sites in the visual thalamus were always within the dLGN, there were sometimes spillover expressions in the neighboring ventral LGN (vLGN), intergeniculate leaflet (IGL), and the pulvinar nucleus similar to previously reported60. The vLGN and IGL do not project to V1 (ref. 61); compared to the dLGN, projections from the pulvinar to V1 are much sparser and limited to the superficial L1 (ref. 62). Hence, the majority of thalamic axons that we imaged in V1 most likely originated from the dLGN.

Retrograde viral labeling

To achieve GCaMP6 expression in V1 neurons projecting to area RL or PM we employed a slightly modified surgery protocol. First, we implanted a custom head-fixing imaging head-plate and mapped the visual cortex using ISOI through the intact skull. This allowed us to identify the precise anatomical location of areas RL and PM. Next, we performed a single, local virus injection into either area RL or PM by slowly injecting 100 nl of ssAAV-retro/2-hSyn1-mRuby2-GCaMP6m-WPRE (7 × 1012 vg/ml, VVF Zurich #v187-retro) at depths of 100−300 µm. This AAV-retro serotype permits selective retrograde labeling of projection neurons and enables sufficient expression for functional two-photon calcium imaging63. After slowly retracting the micropipette, the craniotomy was carefully cleaned and the exposed skull covered with a silicone sealant (Kwik-Cast, World Precision Instruments). One day later, the animal was implanted with a chronic cranial window, exposing V1 for two-photon calcium imaging targeted to V1 neurons projecting specifically to either the RL or PM area.

Diphtheria toxin injections

To abolish retinal direction selectivity acutely in adult mice, we injected diphtheria toxin intravitreously into ChAT-Cre × LSL-DTR mice25. Diphtheria toxin stock solution was made from diphtheria toxin (Sigma, D0564) dissolved in phosphate-buffered saline (PBS) to a concentration of 1 µg/µl and stored at −80 °C. Before injections, the stock solution was diluted in PBS to a final concentration of 0.8 ng/µl. Mice were first anesthetized with an intraperitoneal injection of a fentanyl (0.05 mg/kg body weight; Hameln), midazolam (5.0 mg/kg body weight; Hameln) and medetomidine (0.5 mg/kg body weight; Domitor, Orion) mixture dissolved in saline. A hole was made near the border between the sclera and the cornea using a 30-gaugle needle; 2 µl toxin was then injected into the vitreous of both eyes using a borosilicate glass micropipette connected to a pressure injection system (Picospritzer III, Parker). Mice were returned to their home cage after the anesthesia was reversed with an intraperitoneal injection of a flumazenil (0.5 mg/kg body weight; Hameln) and atipamezole (2.5 mg/kg body weight; Antisedan, Orion Pharma) mixture dissolved in saline. Each eye was re-injected 2 days after the initial injection. OMR recordings were performed 7−9 days after the initial injection, and in-vivo two-photon calcium imaging experiments were initiated 10−12 days after the initial injection.

Optomotor response measurement

For recording the optomotor reflex the mouse was placed on a central, raised platform and presented visual stimuli in the form of drifting sinusoidal gratings projected onto a virtual cylinder on the four surrounding computer screens64. The gratings were drifting horizontally at 12°/s, alternating the drift direction every 60°. One trial consisted of six 1 min repeats; after each repeat, the spatial frequency of the stimulus was sequentially changed (0.05, 0.1, 0.2, 0.25, 0.3, 0.4 cycles/°). Mouse head movements were tracked using OKR arena software64, where the angle of the head is automatically calculated and used to quantify the OMR for each stimulus condition64. OMR was determined by calculating the ratio of the sum of frames where head movements occurred in the stimulus direction versus in the opposite direction64.

Cortical two-photon calcium imaging

Imaging was performed 2−4-weeks after virus injections, when most neurons exhibited cytosolic-only GCaMP6 expression. Mice were anesthetized with 0.3–0.8% (typically 0.5%) isoflurane, and chlorprothexine was delivered intraperitoneally (2.5 mg/kg body weight; Sigma) as a sedative12. The stimulated contralateral eye was kept lubricated by hourly application of a thin layer of silicone oil (OFNA Racing, 10,000 molecular weight). Core body temperature was maintained at 37−38 °C using a feedback-controlled heating pad (World Precision Instruments, ATC2000). A subset of experiments was performed in awake mice. To habituate the mice to handling and experimental conditions, each mouse was head-fixed onto the imaging stage with its body restrained in a cylindrical cover, reducing struggling and substantial body movements60,65. The habituation procedure was repeated for 3 days for each mouse at durations of 15, 30, and 60 min on day 1, day 2, and day 3, respectively. Mice were rewarded with chocolate paste (Nutella) at the end of each habituation/imaging session. For imaging, the mouse was placed under the microscope 10 cm from the 47.65 × 26.87 cm (width × height) screen, with the screen subtending 134.46° in azimuth and 102.75° in elevation and angled 30° from the mouse’s midline. The visual area targeted for two-photon calcium imaging was localized based on superimposing the ISOI border map onto the cortical surface. Imaging was performed 50–100 µm (L1), 120–250 µm (L2/3), and 350–550 µm (L4) below the dura using a scanning microscope (VivoScope, Scientifica) with a 7.9 kHz resonant scanner running SciScan version 1.3 with dispersion-compensated 940 nm excitation provided by a mode-locked Ti:Sapphire laser (MaiTai DeepSee, Spectra-Physics) through either a Nikon 16× (0.8 NA; somata imaging) or an Olympus 25× (1.05 NA; axonal bouton imaging) objective. Clear ultrasound gel was used as an immersion medium (Aquasonic, Parker Laboratories). To prevent light leak originating from the visual stimulation, an imaging well was constructed from a black O-ring and the objective shielded with black tape. Average excitation power after the exit pupil of the objective varied from 25 to 60 mW. Typical images had 512 × 512 pixels, at 0.3−0.35 µm per pixel for axons, and 0.92 µm per pixel for somata, and were acquired at 30.9 Hz using bidirectional scanning. By correcting for any slow drifts in neuron or axon location within the field of view using a reference image6,23, we were able to record from the same population of neurons or axons over extended periods of time (∼40 min), allowing us to assess responses as a function of TF conditions. There was no evidence of GCaMP6 bleaching during experiments. Each mouse was imaged repeatedly over the course of 1–2 weeks.

Visual stimuli for cortical two-photon calcium imaging

Visual stimulation for cortical two-photon calcium imaging was generated and presented via Python-based custom-made software. To measure directional tuning, we presented 100% contrast black and white sinusoidal drifting gratings. Drifting gratings were presented in six trials for 3 s at a time, with 3 s of gray screen between presentations, and were drifted in 12 different directions (0°, 30°, 60°, 90°, 120°, 150°, 180°, 210°, 240°, 270°, 300°, and 330°) in a pseudorandomized order, with a spatial frequency of 0.03 cycles/° and TFs of 0.3, 0.75, 1.2, and 1.8 Hz.

Image analysis for cortical two-photon calcium imaging

Imaging data were excluded from analysis if motion along the z-axis was detected. Raw images from somata imaging were corrected for in-plane motion via a correlation-based approach in Matlab55. Raw images from axonal bouton imaging were corrected for in-plane motion using a piecewise non-rigid motion correction algorithm66. ROIs were drawn in ImageJ (Cell Magic Wand; https://github.com/fitzlab/CellMagicWand) and selected based on mean and maximum fluorescence images56: somata ROIs were polygonal; axonal bouton ROIs were circular. The same ROI set was used for all imaging stacks acquired in a given field of view. Fluorescence time courses were computed as the mean of all pixels within ROIs and were extracted using MIJ (http://bigwww.epfl.ch/sage/soft/mij/). Baseline-normalized fluorescence time courses (∆F/F 0 ) were computed using a 60 s 10th percentile filter and 0.01 Hz low-pass Butterworth filter to define F 0 (ref. 56). For each of the twelve directions, the response amplitude in each trial was determined by sorting all ∆F/F 0 values (down-sampled to 15.4 Hz) during the 3 s drift period, and taking the mean of the larger 50% of data points25. Somata and axonal boutons were defined as visually responsive if ∆F/F 0 in the preferred direction of motion exceeded 0.06 (refs. 7,67) in at least one of the four TFs. They were defined as DS if: (1) They were visually responsive; and (2) their DSI exceeded 0.3 (refs. 26,56) in at least one of the four TFs:

$${\mathrm{DSI = }}\frac{{R_{{\mathrm{pref}}}-R_{{\mathrm{opp}}}}}{{R_{{\mathrm{pref}}}{\mathrm{ + }}R_{{\mathrm{opp}}}}}$$

where R pref denotes the mean ∆F/F 0 response to the preferred direction of motion and R opp the mean ∆F/F 0 response to the opposite direction. The preferred direction of motion for each cell was calculated as the angle, in polar coordinates, of the vector sum27,68:

$${\uptheta} = \tan ^{ - 1}\left( {\frac{{\mathop {\sum }

olimits_{i{\mathrm{ = 1}}}^{{\mathrm{12}}} R_i\;{\mathrm{sin}}d_i}}{{\mathop {\sum }

olimits_{i{\mathrm{ = 1}}}^{{\mathrm{12}}} R_i\;{\mathrm{cos}}d_i}}} \right)$$

where d i denotes the motion direction of direction i and R i the mean ∆F/F 0 response to direction i.

OSI was computed as:

$${\mathrm{OSI = }}\frac{{R_{{\mathrm{pref}}}-R_{{\mathrm{orth}}}}}{{R_{{\mathrm{pref}}}{\mathrm{ + }}R_{{\mathrm{orth}}}}}$$

where R pref denotes the mean ∆F/F 0 response to the preferred orientation and R orth the mean ∆F/F 0 response to the orthogonal orientation. The preferred orientation was defined as the axis including the preferred direction and its opposite direction.

Data decomposition and segmentation

To correlate the TF-dependent response properties in individual cortical DS cells and the fractional changes of neurons between control and Frmd7tm mice, we performed decomposition and segmentation of datasets summarizing response features of the identified DS cells. First, we composed a response matrix for each visual area (e.g., area RL) using a total of eight parameters for each neuron: peak ∆F/F 0 amplitudes and DSI values under each of the four TF conditions. The response matrix included datasets from both control and Frmd7tm mice. For the V1 L2/3 response matrix, we pooled the datasets from target-unspecific, PM-projecting, and RL-projecting DS cells. Next, the response matrix was decomposed into two dimensions by PCA. The resulting PCA distributions showed a distribution trend depending on the TF preference of the individual neurons, indicating that neurons sharing the same TF preference tended to be clustered in the local region of the PCA distribution. To quantify the localization of neurons, we segmented the PCA distribution by 8 × 8 grids. We then calculated the fraction of neurons located within each of the grids, and examined the fractional changes between control and Frmd7tm mice. Based on the fractional changes, we statistically classified neurons into three groups: (1) “Increased”, (2) “Decreased”, and (3) “Unchanged” in Frmd7tm mice, compared to control mice. We tested the number of grids for this segmentation, and confirmed that the results were not qualitatively changed by the size of the grids (Supplementary Fig. 6).

To investigate the relationship between the effects of altered retinal horizontal direction selectivity and the target region of V1-projecting DS cells, we analyzed the fraction of PM- and RL-projecting cells in each grid based on a projection target index (PTI):

$${\mathrm{PTI}}^i{\mathrm{ = }}\frac{{F_{{\mathrm{RLp}}}^i-F_{{\mathrm{PMp}}}^i}}{{F_{{\mathrm{RLp}}}^i + F_{{\mathrm{PMp}}}^i}}$$

Where \(F_y^x\) denotes the fraction of cells projecting to area y in a grid x. A positive PTI indicates that the neurons within the grid are biased towards RL-projecting neurons, while a negative PTI indicates a bias towards PM-projecting neurons.

The effects of altered retinal horizontal direction selectivity was evaluated using a mutation index (MI):

$${\mathrm{MI}}^i = \frac{{F_{{\mathrm{Control}}}^i-F_{{\mathrm{Frmd7tm}}}^i}}{{F_{{\mathrm{Control}}}^i + F_{{\mathrm{Frmd7tm}}}^i}}$$

Where \(F_y^x\) denotes the fraction of cells in population y in grid x. A positive MI indicates that the fraction of neurons originating from Frmd7tm mice is decreased in the grid, while a negative MI indicates that the fraction is increased.

Virus injections for retinal two-photon calcium imaging

For intravitreal viral injections, mice were anesthetized with an intraperitoneal injection of a fentanyl (0.05 mg/kg body weight; Hameln), midazolam (5.0 mg/kg body weight; Hameln), and medetomidine (0.5 mg/kg body weight; Domitor, Orion) mixture dissolved in saline. We made a small hole at the border between the sclera and the cornea with a 30-gauge needle. Next, we loaded 2 µl of AAV1-CAG-GCaMP6s-WPRE-SV40 (1 × 1013 vg/ml, Penn Vector Core, #AV-1-PV2833) into a pulled borosilicate glass micropipette, and the AAV was pressure-injected through the hole into the vitreous of the left eye using a Picospritzer III (Parker). Mice were returned to their home cage after anesthesia was reversed by an intraperitoneal injection of a flumazenil (0.5 mg/kg body weight; Hameln) and atipamezole (2.5 mg/kg body weight; Antisedan, Orion Pharma) mixture dissolved in saline.

Retinal two-photon calcium imaging

Retinal imaging was performed 3−4-weeks after virus injections. Mice were first dark-adapted for 1 h, and next the retina was prepared68. The retina was isolated from the left eye, and mounted ganglion-cell-side up on a small piece of filter paper (Millipore, MF-membrane), in which a 2 × 2 mm aperture window had previously been cut. During the procedure, the retina was illuminated by dim red light (KL1600 LED, Schott) filtered with a 650 nm high-pass optical filter (650/45×, Chroma) and bathed in extracellular solution (in mM): 110 NaCl, 2.5 KCl, 1 CaCl 2 , 1.6 MgCl 2 , 10 d-glucose, 22 NaHCO 3 bubbled with 5% CO 2 , 95% O 2 . The retina was kept at 35−36 °C and continuously superfused with oxygenated extracellular solution during recordings. For retinal two-photon calcium imaging we employed an equipment setup similar to that previously employted68. The isolated retina was placed under a microscope (SliceScope, Scientifica) equipped with a galvo-galvo scanning mirror system (8315 K, Cambridge Technologies), a mode-locked Ti:Sapphire laser tuned to 940 nm (MaiTai DeepSee, Spectra-Physics), and an Olympus 20× (1.0 NA) objective. The GCaMP6s signals emitted were passed through a set of optical filters (ET525/50 m, Chroma; lp GG495, Schott) and collected using a GaAsP detector (Scientifica). Images were acquired at 6−10 Hz using custom-made software developed by Zoltan Raics (SELS Software).

Visual stimuli for retinal two-photon calcium imaging

The visual stimulation was generated via custom-made software (Python and LabVIEW) developed by Zoltan Raics, projected by a DLP projector (LightCrafter Fiber E4500 MKII, EKB Technologies) coupled via a liquid light guide to an LED source (4-wavelength high-power LED Source, Thorlabs) with a 400 nm LED (LZ4-00UA00, LED Engin) through a band-pass optical filter (ET405/40×, Chroma), and focused onto the photoreceptor layer of the mounted retina through a condenser (WI-DICD, Olympus). The stimuli were exclusively presented during the fly-back period of the horizontal scanning mirror68. To measure directional tuning and TF preference, we presented 100% contrast black and white sinusoidal drifting gratings (mean intensity, 0.058 mW/cm2). Light intensity was measured using a power meter (PM200, Thorlabs) and a spectrometer (USB4000-XR1, Ocean Optics). Drifting gratings were presented in 3 trials for 3 s at a time, with 3 s of gray screen between presentations, and shown in 8 different directions (0°, 45°, 90°, 135°, 180°, 225°, 270°, and 315°) in a pseudorandomized fashion, with a spatial frequency of 0.03 cycles/° and TFs of 0.3, 0.75, 1.2, and 1.8 Hz. To measure ON and OFF responses, we presented static flash spots (2 s in duration, 50, 100, 200, 400, 800 µm in diameter). To classify retinal cells into ON-OFF and non-ON-OFF populations, we used an ON-OFF index (OOI)25 (Supplementary Fig. 9):

$${\mathrm{OOI = }}\frac{{R_{{\mathrm{ON}}}-R_{{\mathrm{OFF}}}}}{{R_{{\mathrm{ON}}}{\mathrm{ + }}R_{{\mathrm{OFF}}}}}$$

Where R ON and R OFF denote peak calcium responses during the static spot illumination phase, and the phase after the illumination, respectively. If the mean OOI for 50−800 µm spots was <0.3, the cell was defined as an ON-OFF cell. We calculated the DSI for individual cells, and defined cells with DSI > 0.3 as DS cells, similar to experiments performed in the cortex.

Image analysis for retinal two-photon calcium imaging

The raw two-photon scanning imaging data acquired was initially loaded into Matlab and converted into accessible image files. The ROIs for cell bodies of retinal cells were drawn in Matlab by fitting polygons, and selected based on mean and maximum fluorescence images. Fluorescence time courses were computed as the mean of all pixels within the ROI at each timepoint and were extracted in Matlab. The raw GCaMP6 fluorescence signals for each ROI were normalized (∆F/F 0 ) using the mean fluorescence (F 0 ) in a 2 s window before visual stimulation, and then synchronized with visual stimulus information. The ∆F/F 0 signals were resampled using the interp function in Matlab, and smoothed by a moving average filter (width: two data points). To evaluate cell responsiveness, we determined a threshold for each cell as mean ∆F/F0 + 2 SD ∆F/F0 , and any cell with response amplitudes higher than their threshold was defined as visually responsive and included in further analysis.

Histology and confocal imaging

To validate the injection site in the dLGN, mice were anesthetized with an intraperitoneal injection of a fentanyl (0.05 mg/kg body weight; Hameln), midazolam (5.0 mg/kg body weight; Hameln) and medetomidine (0.5 mg/kg body weight; Domitor, Orion) mixture dissolved in saline, and transcardially perfused with PBS and then with 4% paraformaldehyde (PFA). Brains were removed, fixed overnight in PFA and then transferred to PBS and stored at 4 °C. Brain slices (200 µm thick) were collected in the coronal plane using a vibratome (Leica, VT1000S). Slices were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; 1:1000 dilution, ThermoFisher) before mounting with mounting medium (Fisher Scientific). Images of 1024 × 1024 pixels were acquired using a confocal microscope (Zeiss LSM 780) with a 10× (0.45 NA) objective. To validate the specificity of starburst amacrine cell ablation in diphtheria toxin-injected ChAT-Cre × LSL-DTR mice and PBS-injected littermates, we performed immunohistochemical analyses of the retinas27. A nasal mark was applied to the eyes and fixed for 20 min at room temperature (RT) in 4% PFA before dissection. Afterwards, eyes were rinsed in PBS, dissected, mounted on flatmount paper in 4% PFA for 30 min at RT and then washed with PBS overnight at 4 °C on a shaker. The next day, retinas were incubated in 30% sucrose in PBS for at least 3 h at RT. Afterwards, retinas were transferred in the sucrose buffer to microscope slides (SUPERFROST PLUS, Thermo Scientific) and frozen and thawed three times using dry ice to enhance antibody penetration. After washing with PBS, retinas were blocked for 3 h in blocking buffer (1% bovine serum albumin (BSA), 10% normal donkey serum (NDS), 0.5% TritonX 100, 0.02% sodium azide in PBS) at RT. Primary antibodies (rabbit anti-RBPMS 1:500 [Milipore, ABN1362] and goat anti-ChAT 1:200 [Milipore, ABN1144P]) were incubated for 5 days in antibody reaction buffer (1% BSA, 3% NDS, 0.5% TritonX 100, 0.02% sodium azide in PBS) at 4 °C on a shaker. Secondary antibodies (donkey anti-rabbit IgG Alexa Fluor 568 1:200 [Invitrogen], donkey anti-goat IgG Alexa Fluor 488 1:200 [Life Technologies] together with DAPI 1:1000 [ThermoFisher]) were incubated overnight at 4 °C in antibody reaction buffer. After a final wash in PBS, retinas were embedded in Fluoromount-G (eBioscience). For cell density analysis, z-stacks containing images of 1024 × 1024 pixels (1.38 µm per pixel) were acquired at an interval of 4 µm (total thickness of 75−80 µm) with a confocal microscope (Zeiss LSM 780) using a 10× (0.45 NA) objective, and cells were counted using ImageJ. For detailed confocal z-stacks, we used a 40× (1.4 NA) objective and acquired 1024 × 1024 pixel (0.35 µm per pixel) images at an interval of 0.3 µm (total thickness of ∼75 µm).

Statistical analysis

Statistical tests were performed in Matlab and we used the following statistical tests where appropriate: Mann–Whitney U-test, Wilcoxon signed-rank, Kolmogorov–Smirnov, and χ2 with Yates correction. Rayleigh’s test for non-uniformity of circular data was performed using the Circular Statistics Toolbox69. No testing was performed to check for normality or homogeneity of variance. Center and spread values are reported as mean ± SEM. We used no statistical methods to plan sample sizes, but used sample sizes similar to those frequently used in the field6,56. The number of animals and cells is included in the text or in figure legends. We did not use any randomization; data collection and analysis were not performed blind to the conditions of the experiments. No collected data were excluded from analysis. P-values <0.05 were considered to be statistically significant. When a statistical test was used, the P-value is noted either in the manuscript text or depicted in figures and legends as: *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant, P ≥ 0.05.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.