Image motion with color contrast suffices to elicit an optokinetic reflex in Xenopus laevis tadpoles

The optokinetic reflex is a closed-loop gaze-stabilizing ocular motor reaction that minimizes residual retinal image slip during vestibulo-ocular reflexes. In experimental isolation, the reflex is usually activated by motion of an achromatic large-field visual background with strong influence of radiance contrast on visual motion estimation and behavioral performance. The presence of color in natural environments, however, suggests that chromatic cues of visual scenes provide additional parameters for image motion detection. Here, we employed Xenopus laevis tadpoles to study the influence of color cues on the performance of the optokinetic reflex and multi-unit optic nerve discharge during motion of a large-field visual scene. Even though the amplitude of the optokinetic reflex decreases with smaller radiance contrast, considerable residual eye movements persist at the ‘point of equiluminance’ of the colored stimuli. Given the color motion preferences of individual optic nerve fibers, the underlying computation potentially originates in retinal circuits. Differential retinal ganglion cell projections and associated ocular motor signal transformation might further reinforce the color dependency in conceptual correspondence with head/body optomotor signaling. Optokinetic reflex performance under natural light conditions is accordingly influenced by radiance contrast as well as by the color composition of the moving visual scene.


Material and methods
Animals. Experiments were performed in vitro on semi-intact preparations of Xenopus laevis tadpoles (n = 46) and complied with the "Principles of animal care", publication No. 86-23, revised 1985 of the National Institute of Health. All experiments were carried out in accordance with ARRIVE guidelines and regulations. Permission for these experiments was granted by the ethics committee for animal experimentation of the legally responsible governmental institution (Regierung von Oberbayern) under the license code 55.2-1-54-2532.3-59-12. In addition, all experimental methods were performed in accordance with the relevant guidelines and regulations of the Ludwig-Maximilians-University Munich. Xenopus larvae of either sex at stage 51-54 25 were obtained from the in-house animal breeding facility at the Biocenter-Martinsried of the Ludwig-Maximilians-University Munich. Animals were maintained in tanks with non-chlorinated water (17-18 °C) at a 12/12 light/ dark cycle prior to experimentation. Experimental approach. Semi-intact preparations were obtained according to the procedure reported previously 10,26 . In brief, tadpoles were anesthetized in 0.05% 3-aminobenzoic acid ethyl ester (MS-222; Pharmaq) in frog Ringer solution (in mM: 75 NaCl, 25 NaHCO 3 , 2 CaCl 2 , 2 KCl, 0.5 MgCl 2 and 11 glucose, pH 7.4) and decapitated. For experiments that employed eye motion recordings, the skin covering the dorsal head was removed, the soft skull tissue opened and the forebrain disconnected. This surgical procedure anatomically and functionally preserved the remaining CNS with the eyes and associated optic nerve, extraocular motor innervation and eye muscles. Such preparations allowed prolonged experimentation and in vivo-like activation of the OKR by horizontal large-field visual image motion under defined in-vitro conditions 10,12 . For electrophysiological recordings of RGC axons in these preparations, the optic nerve of the right eye was cleaned from surrounding connective tissue and transected before entering the optic chiasm. All extraocular muscles of this eye were transected at their proximal insertion site to immobilize the eye in its natural position within the head. After the surgery, all preparations were allowed to recover from the surgical intervention for three hours 27 . Eye motion capture and optic nerve recordings. Semi-intact preparations were mechanically secured with insect pins to the Sylgard floor of a Petri dish (5 cm in diameter). As described earlier 12 , the chamber, which was constantly perfused with oxygenated frog Ringer solution at a rate of 3.0-5.0 ml/min, was placed in the center of an open cylindrical screen with a height of 5 cm and a diameter of 8 cm, encompassing a horizontal visual field of 275°. Three digital light processing (DLP) video projectors (Aiptek V60), installed at 90° angles to each other projected visual motion stimuli onto the screen 12,28 at a refresh rate of 60 Hz. For eye motion recordings, a CCD camera (Grasshopper 0.3 MP Mono FireWire 1394b, PointGrey, Vancouver, BC, Canada), mounted 20 cm above the center of the recording chamber, permitted on-line tracking of horizontal eye movements by custom-written software 29 . The position of both eyes was digitized at a sampling rate of 50 Hz and recorded along with the visual motion stimulus (Spike2 version 7.04, Cambridge Electronic Design Ltd., Cambridge, United Kingdom). The chamber was illuminated from above using an 840 nm infrared light source. An infrared longpass filter inside the camera ensured selective transmission of the respective wavelengths and a high contrast to outline the eyeballs for motion tracking and online analysis of induced eye movements.
Electrophysiological recordings of multi-unit optic nerve spike activity were performed under the same experimental conditions as described previously 10 . In brief, the spike discharge of retinal ganglion cells was recorded extracellularly (EXT 10-2F, npi Electronics, Tamm, Germany) with glass microelectrodes that were filled with Ringer solution 10 . Electrodes were produced with a horizontal puller (P-87 Brown/Flaming, Sutter Instruments Company, Novato, CA, USA) and the tips were broken and individually adjusted to fit the diameter of the transected optic nerve 10

Stimulus paradigms.
A reliable estimate of color motion perception is provided by the performance of visuo-ocular motor responses evoked by moving chromatic and radiance contrast stimuli 12 . This method depends on the brightness ratio of two colors at which the response is minimal 30,31 . To identify this ratio, animals were presented with horizontal visual image motion stimuli using a vertically striped pattern with alternating red and blue vertical stripes. With our digital projection system, the red and blue color provided the maximal possible separation of the wavelength spectrum (Fig. 1B). The intensity of the red stripes was varied systematically, leading to variations in the OKR response due to resulting differences in radiance contrast. At the point of equiluminance (POE), the respective brightness of the optic scene appears to be homogeneous to the animal, such that the visual image is only structured by the color of the scenery. At the POE, a visuo-motor response can  www.nature.com/scientificreports/ either be absent, indicative of a color-blind motion perception system or show a residual response, indicating that color information provides motion cues 12 . Here, we exploited the robust OKR of larval Xenopus 10 that is elicited by horizontal motion of a large-field visual scene ( Fig. 1A1-4) with a rectangular velocity profile of ± 10°/s and a frequency of 0.2 Hz.
Point of equiluminance. The POE was determined by presenting a visual stimulus that consisted of alternating red and blue vertical chromatic stripes (Fig. 1A4). The radiance of the red stripes was varied over a range that extended from 0.29 to 3.18 W*sr −1 m −2 , while the intensity of the blue stripes was maintained at a constant value of 4.66 W*sr −1 m −2 . As a control condition to determine the amplitude of spontaneous eye oscillations or measurement noise, the amplitude of eye movements at the stimulus frequency, while presenting a uniformly lit grey screen, served as reference level for comparison of the minimum OKR amplitude (Grey condition, radiance 3.21 W*sr −1 m −2 ). Importantly, the POE was determined individually for each animal as intensity value of the red stripes at which the OKR response was minimal. The mean POE was constructed by averaging the separately obtained values of each animal.
Interaction of color and motion at high light intensities. Three visual motion stimuli were presented at high intensity levels in random order to assess the interaction of color and radiance motion at high contrast levels. Stripes were white (18.7 W*sr −1 m −2 ), red (3.39 W*sr −1 m −2 ), blue (4.66 W*sr −1 m −2 ) or black (0.187 W*sr −1 m −2 ) and were presented in alternating combinations of white/black, red/black, blue/black and red/blue stripes ( Fig. 1A1-4).
Retinal ganglion cell discharge at high light intensities. The encoding of colored motion stimuli at high intensities at the neuronal level was determined by recordings of the optic nerve discharge during horizontal motion of a large-field visual scene. The motion stimulus had a sinusoidal profile with a peak velocity of ± 10°/s and a frequency of 0.125 Hz causing similar positional stimulus oscillations as those used to evoke eye movements.

Retinal ganglion cell discharge near the POE.
To determine whether the discharge of retinal ganglion cells is exclusively driven by radiance contrast, or whether particular units also respond to pure color contrast, the spike activity was determined in response to red/blue stripes at three different red color intensities close to the POE (1.14, 1.19 and 1.25 W*sr −1 m −2 ), while the intensity of the blue stripes was kept constant at 4.66 W*sr −1 m −2 .
Spectral distribution of color stimuli. Individual red and blue colors were generated using the red and blue channels of the image projectors; white stripes were generated using all three color channels (for spectra see Fig. 1B,C). This procedure was chosen for the following reason: if red-black or blue-black stimuli would cause larger responses than white-black stimuli, then the neuronal transformation of RGC signals into optokinetic eye movements must depend on specific chromatic contrasts rather than pure radiance/luminance responses. Thus, if, for example, red-responsive RGCs would be exclusively responsible for eliciting the OKR, then it can be expected that the response to white-black stripes is equal or even stronger than red-black stripes, because redresponsive RGCs would be activated even better with white light (see spectral composition in Fig. 1B). Spectra and radiance values were measured using a spectrometer (PhotoResearch SpectraScan PR655).

Data analysis.
To obtain a robust measure of the strength of the optokinetic response, the OKR magnitude was computed by fitting a triangular profile to the recorded eye position trace and evaluating the amplitude of the fit 12 . To account for the fact that the "actual" value of the POE might lie between the sampled responses at different intensities, the resulting intensity-amplitude curve was fitted with a function for the normalized OKR amplitude A defined by: with the subjective brightness for red or blue: L Red and L Blue represent the radiance values for red and blue and c Red and c Blue are the relative sensitivities to red and blue, respectively. The parameters m and c were required to model the individual sensitivity of each preparation's optokinetic response to changes in contrast. This model was able to fit the observed data, particularly near the POE. The POE was identified as the radiance value of the red stripes at which the fitted function became minimal (A CR ) individually for each preparation. The chromatic response (CR) component of the optokinetic eye movement was then defined as the OKR amplitude at this point.

Spike sorting.
To determine the response properties of individual retinal ganglion cells, spike sorting was performed in MATLAB (R2017a) on multi-unit optic nerve spike discharge in selected experiments, where individual units were identifiable. For each preparation, all trials were pooled following detection of the action potentials by a non-linear energy operator, the Teager-Kaiser energy operator 32 , with an optimized threshold 33 . Artifacts were identified as all segments of the dataset in which the electrical signal was larger than 1000 mV www.nature.com/scientificreports/ and were subsequently removed by replacing the respective values with zero. Spikes were then extracted in a 7 ms window around their respective peaks (200 samples at a sampling rate of 28.6 kHz). The collected spike shapes were subjected to singular value decomposition. The three largest singular values were then used for spike sorting. The values were clustered using k-means clustering (MATLAB 2017a). The number of clusters was determined by visual inspection of the 3D scatter plot of singular values to ensure that data were clustered into non-overlapping regions. Spike clustering was very clear in some cases and somewhat less clear in others; only clusters that could be clearly distinguished were taken into account for an evaluation of the respective spike activity of usually up to four units. Firing rate magnitude of individual RGCs was determined as the total count of action potentials during each trial. Since all trials had the same duration and velocity profile, this gave a robust estimate of single-unit activity during stimulation with differently colored stripe combinations.
Statistical procedures. The significance value was chosen as α = 0.05 for all statistical tests. The response amplitudes of the OKR at the POE were compared to the reference values obtained from the grey condition using a two-sample t-test. The OKR amplitudes in response to white/black, blue/black, red/black and red/blue vertical stripe motion were compared using a repeated measures ANOVA. As described earlier 12 , post-hoc-tests were performed using paired t-tests between all conditions and the Bonferroni method to compensate for multiple comparisons. To reveal a possible linear interaction between color and radiance motion information, the correlation between the chromatic component of the OKR and the differences between the responses to red/blue, red/ black, blue/black and white/black stimuli was computed. To account for potential mathematical coupling, statistical significance of correlations was assessed by randomly re-sampling data points to estimate the distribution of expected correlation coefficients 12 . To determine the response magnitude of individual optic nerve fibers to colored compared to pure radiance stimuli, color preference (g Red, Blue ), determined as relative increase or decrease of activity in response to colored versus white stimuli was measured by computing the log of the ratio of spike counts (SC) in either red or blue color condition over the spike count in white condition: Presentation. Schemes, data and analysis plots were generated with MATLAB (R2017a) and assembled in

Results
Determination of the point of equiluminance. Large-field visual motion stimulation with black/ white-striped image patterns (Fig. 1A1) triggers robust conjugate ocular motor following responses of both eyes in semi-intact preparations of Xenopus laevis tadpoles ( Fig. 2A) 10 . Horizontally alternating constant-velocity motion stimulation with a black/red-or black/blue-striped pattern (Fig. 2B) elicited eye movements with comparable magnitudes in such preparations. The robustness of these responses allowed determining the POE between the two colors (Figs. 1C, 2C). The POE at which the radiance contrast of the red stripes with respect to blue stripes with a radiance of 4.66 W*sr −1 m −2 vanished was very similar across animals and occurred on average at a value of 1.22 ± 0.09 W*sr −1 m −2 (Fig. 2C), confirming previous findings that the relative sensitivities to these two component colors show little variance between animals 12 . The normalized amplitude of the OKR at the respective individual POE, separately determined for each animal was 0.27 ± 0.15 with respect to the mean amplitude over all conditions (Fig. 2D). This residual eye movement magnitude, albeit small, was significantly greater than baseline oscillations (Two-sampled t-test: t(42) = 3.02, p = 0.004, d = 0.86; Fig. 2D). As a novel finding, this indicated that color contrast is sufficient to detect motion of the visual scene and to consequently evoke an ocular motor response.
Interaction of color and radiance during OKR performance. In addition to the OKR evoked by pure color contrast stimuli, high intensity color/black-striped motion stimuli provoked eye movements with amplitudes that were considerably larger compared to those elicited by a white/black-striped pattern. This is indicated by a significant main effect of stimulus color (Repeated measures ANOVA: F (3, 102) = 46.62, p < 0.001, η 2 = 0.26; Fig. 3A) and successive post-hoc tests. This finding was rather surprising since the radiance of white stripes was approximately four-fold compared to that of the blue and more than five-fold compared to that of red the stripes (18.7 W*sr −1 m −2 compared to 4.66 W*sr −1 m −2 or 3.39 W*sr −1 m −2 , respectively). In contrast, the black stripes had the same intensity. The effect persisted even when the radiance of white and colored stripes was matched (Two-sample t-test for blue: t(5) = − 2.90, p = 0.034, d = − 0.377). The additional color-related components, however, were not significantly correlated with the isolated chromatic OKR response at the POE (blue: ρ = 0.04, p = 0.430; red: ρ = 0.05, p = 0.428; Fig. 3B1,3). Bayes factor analysis 34 showed, however, that there is only anecdotal evidence for a lack of effect of response difference on chromatic response (B 01 = 2.98 for red-black minus whiteblack; B 01 = 2.95 for red-black minus blue-black). The fact that additional color-related components of red and blue motion stimuli were strongly correlated (ρ = 0.71, bootstrap p < 0.011; Fig. 3B2), suggested a particular color motion sensitivity of some but not all preparations. www.nature.com/scientificreports/ Furthermore, comparison between color motion-stimuli revealed significantly larger OKR amplitudes in response to a blue/black-, than to a red/black-striped motion stimulus (Figs. 2B, 3A), indicating that the performance of the OKR in Xenopus tadpoles differs for motion stimuli with different color patterns. Variation of pure radiance contrast of achromatic stimuli as previously employed 10 indicated that beyond a certain contrast level, OKR amplitudes saturate. Based on these previous findings, the high contrast stimuli described above fall into the saturation range and no local sensitivity to changes in contrast is expected. Nevertheless, the increase in OKR amplitude to color stimulus motion at distinctly lower radiance contrasts compared to black/white stimuli suggests the presence of a color-related component that influences the OKR performance.

Optic nerve population responses to colored visual motion stimuli. Electrophysiological record-
ings of the optic nerve with suction electrodes consistently evoked a multi-unit discharge of retinal ganglion cells albeit with bidirectional activity patterns during horizontal stimulus motion ( Fig. 4A and magnified response over one cycle). An earlier study demonstrated for radiance motion stimuli a close association between the www.nature.com/scientificreports/ population activity of the optic nerve and the speed of the large-field visual image motion 10 . This indicates that RGC population activity can be interpreted as surround velocity estimate. Whether this also applies when comparing responses to stimuli with distinct wavelength components-that differentially excite retinal photoreceptors-was evaluated by a set of experiments with the same color combination stimuli as used for evaluating the performance of the OKR. Interestingly, at the population level, the relative neuronal response magnitude of the multi-unit optic nerve discharge (spike count) differed from the behavioral observations (two-sample Kolmogorov-Smirnov test, p < 0.001; compare Fig. 3A with Fig. 4C). Although the neuronal response to a high intensity red/blue-striped pattern was smaller than the response to the three other patterns (likely due to a smaller radiance contrast in this condition; Fig. 4C), the respective response magnitudes showed a different activation pattern compared to the OKR. This suggests that the mechanism that causes different optokinetic response amplitudes for different colors at high light intensities 12 differs from the mechanism responsible for adjusting the optokinetic response simply based on radiance contrast.
Optic nerve single-unit responses to colored visual motion stimuli. Spike sorting of the multiunit optic nerve discharge and isolation of individual units using SVD-based spike sorting (MATLAB R2017a) revealed an alternative mechanism for the partially color-specific optokinetic response amplitude (see units 1-4 in Fig. 4B and corresponding PSTHs in Fig. 5). In fact, the presence of separate subgroups of retinal motion detectors, which respond preferentially to particular wavelengths could be the origin of this color dependency. Mediation of visual motion signals in distinct color channels from the retina to the pretectum and differential coupling probability or strength within the OKR circuitry could explain the observed differential color sensitivity of this reflex.
To test this hypothesis, responses of isolated single optic nerve units (n = 25) that modulated their firing rate with stimulus velocity were analyzed. The color preference was identified by evaluating the response magnitude to the four high intensity color stimuli (Fig. 4C). While the total spike count varied strongly between individual units, clear differences were encountered between the color-preference of the different units, with illustrates the difference in response magnitudes between blue-black and white-black (x-axis) and red-black and white-black (y-axis); despite the mathematical coupling, there is a significant correlation between blue/ white and red/white response magnitudes (expected correlation due to coupling: ρ = 0.48).  www.nature.com/scientificreports/ color preferences ranging between − 0.42 and + 0.59 (10th and 90th percentile, respectively), corresponding to relative responses to colored stimuli between 66 and 181% of the same unit's responses to white stripes. There was a roughly even split between units responding preferentially to either white (n = 12), or colored (red: n = 10, blue: n = 3) stripes. To support the finding that units can be separated into subtypes with preferential sensitivity to broad and narrow light spectra, respectively, Gaussian mixture models with one and two components were fitted to the units' color preference values. Despite the additional degrees of freedom, the two-component model provided higher explanatory value, indicated by a lower Akaike Information Criterion (14.6 versus 16.9; Fig. 4D).
The distinction between units with color-sensitive and color-insensitive response characteristics was obvious and allowed clear identification of a neuronal population preferentially responding to achromatic stimuli. Among color-sensitive units, however, there was no clear distinction based on the response preference to either red or blue color. In contrast to behavioral findings that animals with a strong preference for one color (expressed by their relative OKR amplitude) also show a strong response to the other color, there was no significant correlation between red and blue response preference on the level of RGC axons (ρ = 0.67, p = 0.12). Nevertheless, there was a considerable degree of variability in preference for colored or white stimuli (Fig. 4D). While most of the measured units were inactive during stimulation near the POE (Fig. 4E2), a small number of single units still clearly showed an activity profile that modulated with instantaneous stimulus velocity (see Fig. 4E1 for an example), suggesting that a set of RGC axons encode and transmit color-dependent visual motion signals.

Discussion
The optokinetic reflex of Xenopus laevis tadpoles is systematically influenced by the color component of the moving visual scene. Even though eye movement amplitudes decrease with smaller radiance contrast, a residual optokinetic response at the point of equiluminance remains, even when accounting for inter-individual variance in the exact location of the POE. This color contrast-dependency complies with an activation of larger OKR amplitudes when black stripes were paired with colored instead of white stripes. The underlying neuronal www.nature.com/scientificreports/ computation is likely of retinal origin since single optic nerve fibers can show considerable preferences for either colored or white motion stimuli. The residual response at the POE demonstrated that pure color contrast is a likely contributor to the visual motion detection mechanism, which forms the basis of the optokinetic reflex 10 . In fact, the OKR at POE along with the residual directionally selective response of RGCs (see Fig. 4E1) suggests that retinal motion processing contributes at least in part to the observed behavioral responses. Accordingly, by varying the radiance of the red stripes a point was reached where the behavioral response became minimal. Thus, the response at the POE for red-blue stripes indicates that the motor response is in fact elicited not just by radiance contrast, but also by color contrast. Anatomically, the necessary requirements for color vision have been shown to be present in adult Xenopus that possess principal and thin rods as well as four types of cones with red, blue and ultravioletsensitive opsins 35 . The current study provides evidence that Xenopus tadpoles not only possess but also recruit these anatomical structures during large-field visual motion to distinguish different colors for an adjustment of their ocular motor output. In fact, larval Xenopus can be trained to avoid specifically-colored segments of the environment, concluding that color vision is behaviorally relevant for these animals 36 .
Visual motion-induced behavior at the point of equiluminance facilitates a determination of the dependency of visual motion perception on color 37 . This method has been particularly applied to optomotor responses of animal models, such as flies 31 or zebrafish 38,39 , where motion vision was found to be "color-blind", at variance with earlier reports on amphibians such as frogs 13,14,40 demonstrating that orienting head/body movements in both larvae and adults of ranid frogs are color-sensitive. If this is a fundamental difference between amphibians and zebrafish is likely but so far not yet fully explored. Although visual motion-driven ocular (this study) as well as neck/limb motor behaviors 13,14 are color sensitive in amphibians, the color-sensitivity of the OKR, its shortlatency neuronal circuitry, approximately linear input-output relationship along with the spatial specificity and the limited degrees of freedom of eye movements 41 makes this ocular motor behavior a very sensitive assay. This is further facilitated by the possibility to isolate small responses based on their frequency characteristics and to robustly distinguish evoked reflexive from spontaneous motor activity.
Our experiments revealed a non-zero optokinetic response at the POE in the majority of preparations (26 out of 35), suggesting that color information indeed plays a role in low-level motion vision, even though the central targets of the recorded fibers and functional implication remained so far unidentified. The contribution of color to visual motion processing appears to be independent of the overall light level, provided these RGCs feed into the ocular motor reflex circuitry, since color influences the performance of the OKR also at high light intensities, when alternating white/black, red/black and blue/black stripes were presented. Such a color contribution to neuronal motion computation, however, is not restricted to brainstem levels but is also implemented in higher-order motion vision, such as for human subjective speed estimation 15 . While the speed of an equiluminant chromatic grating is only perceived to be moving at about half the speed of a corresponding luminance grating, a motion percept is still evoked in the absence of luminance contrast 17 .
The larger responses to stimuli combining black with either blue or red stripes, as compared to white/black stripes, was rather surprising, in particular since the radiance contrast of white versus black stripes was the highest. While obviously all tested stimulus patterns express sufficient radiance contrast for maximal optokinetic responses, colored motion stimuli likely recruit additional pathways thereby leading to increased amplitudes of the OKR. Although direct causality is still lacking, this dependency of OKR amplitudes complies with the observed pattern in the population of optic nerve fibers 10 . This indicates that radiance-dependent variations in the performance of the OKR potentially derive from retinal signal processing.
At variance with the correlation between radiance magnitudes and optic nerve population activity 10 , variations of the color composition of visual motion stimuli were generally unrelated to the population activity of retinal ganglion cells (Fig. 4C). This suggests that the observed differences in response amplitude are not caused by radiance contrast artifacts, such as reflections. Rather, the increase of the OKR amplitude during chromatic stimulation could be explained by retinal motion detectors with preferences for specific colors, which differ in how strongly they are coupled to the brainstem OKR circuitry 39 . This hypothesis is supported by our observation of individual units at the optic nerve level with varying preferences for large-field visual motion stimuli in red, blue or white color (Fig. 4B). Evaluating individual units based on how their response changes to white and red or blue stimuli, respectively, showed a relatively large separation between two types (see Fig. 4C). In ~ 50% of the recorded units, the spike activity decreased when color-striped visual motion stimuli were presented, while in the remaining 50% of the examined optic nerve fibers the spike discharge rate increased. This latter augmentation was typically similar for red-and blue-striped motion stimuli, although, units with a preference for either one of the two colors were found. This demonstrates further that a neuronal substrate for color-specific processing of motion information is implemented in the Xenopus as in the zebrafish retina. In the present study, however, it was not possible to clarify whether the analyzed optic nerve fibers were indeed connected via the accessory optic system to the relevant pretectal relay nucleus 4 and thus in fact have contributed to the OKR response. While most of the optic nerve fibers investigated here (examples shown in Fig. 5) did not show a directional preference that would be required to sustain the OKR response (for an exception see Fig. 4E1), their response to both color and motion indicates that color-sensitive motion channels already exist in the amphibian retina and may constitute the origin of color-sensitive OKR responses compatible with the color-sensitivity of optomotor reactions of the head/body 40 .
From an ecological point of view, color information is advantageous for OKR performance. Color provides a number of additional cues about the environment and the visual motion within it and potentially extends the sensitivity of this motor behavior. Color increases the saliency of objects, and thus facilitates motion perception 19 . In addition, certain environmental features have invariant colors and-based thereupon-can be more easily distinguished from other objects. This plays a particular role in the distinction between visual motion of the entire scene (which is most likely caused by self-motion and therefore should elicit an optokinetic ocular motor www.nature.com/scientificreports/ response), and motion of external objects such as floating debris in aquatic environments for instance. One particular example in this environment is a distinction between the blue sky, which always provides a worldstationary reference, in contrast to other environmental features which might be moving themselves, such as the surrounding fauna. Color cues can thus assist in performing a behavioral distinction between different sources of visual motion, in particular self-and object motion.

Data availability
The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.