Author response: Spherical arena reveals optokinetic response tuning to stimulus location, size, and frequency across entire visual field of larval zebrafish
2021-04-08

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Many animals have large visual fields, and sensory circuits may sample those regions of visual space most relevant to behaviours such as gaze stabilisation and hunting. Despite this, relatively small displays are often used in vision neuroscience. To sample stimulus locations across most of the visual field, we built a spherical stimulus arena with 14,848 independently controllable LEDs. We measured the optokinetic response gain of immobilised zebrafish larvae to stimuli of different steradian size and visual field locations. We find that the two eyes are less yoked than previously thought and that spatial frequency tuning is similar across visual field positions. However, zebrafish react most strongly to lateral, nearly equatorial stimuli, consistent with previously reported spatial densities of red, green, and blue photoreceptors. Upside-down experiments suggest further extra-retinal processing. Our results demonstrate that motion vision circuits in zebrafish are anisotropic, and preferentially monitor areas with putative behavioural relevance. Introduction The layout of the retina, and the visual system as a whole, evolved to serve specific behavioural tasks that animals perform to survive in their respective habitats. A well-known example is the position of the eyes in the head which varies between hunting animals (frontal eyes) and animals that frequently need to avoid predation (lateral eyes) (Cronin et al., 2014). Hunting animals keep the prey within particular visual field regions to maximise behavioural performance (Hoy et al., 2016; Bianco et al., 2011; Yoshimatsu et al., 2020). To avoid predation, however, it is useful to observe a large proportion of visual space, especially those regions in which predators are most likely to appear (Smolka et al., 2011; Zhang et al., 2012). The ecological significance of visual stimuli thus depends on their location within the visual field, and it is paralleled by non-uniform processing channels across the retina. This non-uniformity manifests as an area centralis or a fovea in many species, which is a region of heightened photoreceptor density in the central retina and serves to increase visual performance in the corresponding visual field regions. Photoreceptor densities put a direct physical limit on performance parameters such as spatial resolution (Haug et al., 2010; Merigan and Katz, 1990). In addition to these restrictions mediated by the peripheral sensory circuitry, an animal’s use of certain visual field regions is also affected by behaviour-specific neural pathways, for example pathways dedicated to feeding and stabilisation behaviour. The retinal and extra-retinal circuit anisotropies can in turn effect a dependence of behavioural performance on visual field location (Hoy et al., 2016; Bianco et al., 2011; Murasugi and Howard, 1989; Shimizu et al., 2010; Yang and Guo, 2013; Zimmermann et al., 2018; Baden et al., 2013). Investigating behavioural performance limits and non-uniformities can offer insights into the processing capabilities and ecological adaptations of animal brains, especially if they can be studied and quantitatively understood at each processing step. The larval zebrafish is a promising vertebrate organism for such an endeavour, since its brain is small and a wide array of experimental techniques are available (Baier and Scott, 2009; McLean and Fetcho, 2011). Zebrafish are lateral-eyed animals and have a large visual field, which increases during early development and in individual 4-day-old larvae has been reported to cover about 163° per eye (Easter and Nicola, 1996), although little is known about interindividual variability. Their retina contains four different cone photoreceptor types (Nawrocki et al., 1985), each distributed differently across the retina. UV photoreceptors are densest in the ventro-temporal retina (area temporalis ventralis), whereas the red, green, and blue photoreceptors cover more central retinal regions (Zimmermann et al., 2018). Zebrafish larvae perform a wide range of visually mediated behaviours, ranging from prey capture (Trivedi and Bollmann, 2013; Mearns et al., 2020) and escape behaviour (Heap et al., 2018) to stabilisation behaviour (Kubo et al., 2014; Orger et al., 2008); however, the importance of stimulus location within the visual field for the execution of the respective behaviours has only recently been recognized and is still not well understood (Hoy et al., 2016; Zimmermann et al., 2018; Mearns et al., 2020; Kist and Portugues, 2019; Wang et al., 2020; Johnson et al., 2020; Lagogiannis et al., 2020). During visually mediated stabilisation behaviours, such as optokinetic and optomotor responses, animals move their eyes and bodies, respectively, in order to stabilise the retinal image and/or the body position relative to the visual surround. The optokinetic response (OKR) consists of reflexively executed stereotypical eye movements, in which phases of stimulus ‘tracking’ (slow phase) are interrupted by quick phases (Figure 1a). In the quick phases, eye position is reset by a saccade in the direction opposite to stimulus motion. In humans, optokinetic responses are strongest in the central visual field (Howard and Ohmi, 1984). Furthermore, lower visual field locations of the stimulus evoke stronger OKR than upper visual field locations, which likely represents an adaptation to the rich optic flow information available from the structures on the ground in the natural environments of primates (Murasugi and Howard, 1989; Hafed and Chen, 2016). Figure 1 with 2 supplements see all Download asset Open asset Presenting visual stimuli across the visual field. (a) When presented with a horizontal moving stimulus pattern, zebrafish larvae exhibit optokinetic response (OKR) behaviour, where eye movements track stimulus motion to minimise retinal slip. Its slow phase is interrupted by intermittent saccades, and even if only one eye is stimulated (solid arrow), the contralateral eye is indirectly yoked to move along (dashed arrow). (b) Often, experiments on visuomotor behaviour such as OKR sample only a small part of the visual field, whether horizontally or vertically. As different spatial directions may carry different behavioural importance, an ideal stimulation setup should cover all or most of the animal’s visual field. For zebrafish larvae, this visual field can be represented by an almost complete unit sphere. (c) We arranged 232 LED tiles with 64 LEDs each across a spherical arena, such that 14,484 LEDs (green dots) covered nearly the entire visual field. (d) The same individual positions, shown in geographic coordinates. Each circle represents a single LED. Each cohesive group of eight-by-eight circles corresponds to the 64 LEDs contained in a single tile. (e) To identify LED and stimulus locations, we use Up-East-North geographic coordinates: Azimuth α describes the horizontal angle, which is zero in front of the animal and, when seen from above, increases for rightward position. Elevation β refers to the vertical angle, which is zero throughout the plane containing the animal, and positive above. (f) The spherical arena is covered in flat square tiles carrying 64 green LEDs each. (g) Its structural backbone is made of a 3D-printed keel and ribs. Left and right hemispheres were constructed as separate units. (h) Across 85-90% of the visual field, we can then present horizontally moving bar patterns of different location, frequency and size to evoke OKR. Figure 1—source data 1 SCAD files for 3D-printing the arena scaffold. https://cdn.elifesciences.org/articles/63355/elife-63355-fig1-data1-v2.zip Download elife-63355-fig1-data1-v2.zip In zebrafish larvae, OKR behaviour has been used extensively to assess visual function in genetically altered animals (Brockerhoff et al., 1995; Muto et al., 2005; Neuhauss et al., 1999). OKR tuning to velocity, frequency, and contrast of grating stimuli has been measured (Clark, 1981; Cohen et al., 1977; Huang and Neuhauss, 2008; Rinner et al., 2005), and, more recently, zebrafish are used as a model for investigating vertebrate sensorimotor transformations (Kubo et al., 2014; Portugues et al., 2014). While zebrafish can distinguish rotational from translational optic flow to evoke appropriate optokinetic and optomotor responses (Kubo et al., 2014; Naumann et al., 2016; Wang et al., 2019), it is still unclear which regions of the visual field zebrafish preferentially observe in these behaviours. The aquatic lifestyle, in combination with the preferred swimming depths (Lindsey et al., 2010), might cause the lower visual field to contain less relevant information when compared to terrestrial animals. This in turn might result in behavioural biases to other – more informative – visual field regions. A corresponding systematic behavioural quantification in zebrafish or other aquatic species, which would relate OKR behaviour to naturally occurring motion statistics and the underlying neuronal representations in retina and retino-recipient brain structures, has been prevented by technical limitations. Specifically, little is known about (i) the dependence of OKR gain on stimulus location or (ii) on stimulus sizes, (iii) possible interactions between stimulus location, size and frequency, (iv) putative asymmetries between the left and right hemispheres of the visual field, and (v) the relationship between a putative dependence of OKR on stimulus location and zebrafish retinal architecture. In other species with large visual fields, such as Drosophila, full-surround stimulation setups have been designed and used successfully (Reiser and Dickinson, 2008; Kim et al., 2017; Maisak et al., 2013), but to date, none has been used for fish. This is at least partly due to their aquatic environment and the associated difficulties regarding the refraction of stimulus light at the air-water interface. Such distortions of shape can be partially compensated by pre-emptively altering the shape of the stimulus. However, using regular computer screens or video projection, the resulting luminance profiles remain anisotropic unless compensated, potentially biasing the response toward brighter locations. Additionally, most stimulus arenas cannot easily be combined with the recording of neural activity, for example, via calcium imaging, as stimulus light and calcium fluorescence overlap in both the spectral and time domains. These challenges must be overcome to enable full-field visual stimulation in zebrafish neurophysiology experiments (Figure 1b). At least one existing solution for stimulating and tracking freely moving zebrafish supports unusually large stimuli, although despite its versatility, it still only covers part of the visual field and does not address the remaining issues of total internal reflection and the interoperability with laser-scanning microscopes (Stowers et al., 2017 ). Here, we present a novel visual stimulus arena for aquatic animals, which covers almost the entire surround of the animal, and use it to characterise the anisotropy of the zebrafish OKR across different visual field locations as well as the tuning to stimulus size, spatial frequency and leftside versus rightside stimulus locations. We find that the OKR is mostly symmetric across both eyes and driven most strongly by lateral stimulus locations. These stimulus locations approximately correspond to a retinal region of increased photoreceptor density. By rotating the experimental setup and/or the animal, our control experiments revealed that additional extra-retinal determinants of OKR drive exist as well. Our characterisation of OKR drive across the visual field will help inform bottom-up models of the vertebrate neural pathways underlying the optokinetic response and other visual behaviour. Results Spherical LED arena allows presentation of stimuli across the visual field By combining 3D printing with electronic solutions developed in Drosophila vision research, we constructed a spherical stimulus arena containing 14,848 individual LEDs covering over 90% of the visual field of zebrafish larvae (Figure 1c, Materials and methods section on coverage). Using infrared illumination via an optical pathway coupled into the sphere (Figure 1—figure supplement 1a–b), we tracked eye movements of larval zebrafish during presentation of visual stimuli (Dehmelt et al., 2018). To avoid stimulus aberrations at the air-to-water interface, we designed a nearly spherical glass bulb containing fish and medium (Figure 1—figure supplement 1c–d). With this design, stimulus light from the surrounding arena is virtually not refracted (light is orthogonal to the air-to-water interface) and reaches the eyes of the zebrafish larva in a straight line. Thus, no geometric corrections are required during stimulus design (Source code 1), and stimulus luminance is expected to be nearly isotropic across the visual field. We additionally designed the setup to minimise visual obstruction and developed a new embedding technique to immobilise the larva at the tip of a narrow glass triangle (see Materials and methods). In almost all possible positions, fish can thus perceive stimuli without interference (Wang et al., 2021). The distance between most of the adjacent LED pairs is smaller than the photoreceptor spacing in the larval retina (Haug et al., 2010; Tappeiner et al., 2012), resulting in a good spatial resolution across the majority of the spherical arena surface (Figure 1—figure supplement 2, see Materials and methods section on resolution). As flat square LED tiles cannot be perfectly arranged on a spherical surface (Supplementary file 1A), small triangular gaps are unavoidable. More importantly, several gaps in LED coverage, resulting from structural elements of the arena, were restricted mainly to the back, the top, and bottom of the animal. The ‘keel’ behind and in front of the fish supports the horizontal ‘ribs’, and the circular openings in the top and bottom accommodate the optical path for eye tracking or scanning microscopy (also see discussion of arena geometry in our data repository [Dehmelt et al., 2020; https://gin.g-node.org/Arrenberg_Lab/spherical_arena]). Stimulus position dependence of the optokinetic response Horizontally moving vertical bars reliably elicit OKR in zebrafish larvae (Beck et al., 2004). We used a stimulus which rotated clock- and counter-clockwise with a sinusoidal velocity pattern (velocity amplitude 12.5 degree/s, frequency of the velocity envelope 0.1 Hz, spatial frequency 0.06 cycles/degree, Figure 2a). OKR performance was calculated by measuring the amplitude of the resulting OKR slow-phase eye movements after the saccades had been removed (Figure 2b, Figure 2—figure supplement 1, Source code 1C, Materials and methods). The OKR gain then corresponds to the speed of the slow-phase eye movements divided by the speed of the stimulus (which is equivalent to the ratio of the eye position and stimulus position amplitudes). In addition to traditional full-field stimulation, our arena can display much smaller stimuli in different parts of the visual field. These novel stimuli evoked reliable OKR even at remote stimulus locations (Figure 2c, Figure 2—figure supplement 2), and thus allowed us to investigate previously inaccessible behavioural parameters. We excluded any trials from data analysis that showed other behaviours (e.g. drifts and ongoing spontaneous eye movements) superimposed on OKR (Figure 2—figure supplement 2). In addition to the characteristic eye traces (Figure 2c), Bode plots of the magnitude and phase shift relative to the stimulus qualitatively match previously reported zebrafish response to full-field stimulation (Figure 5—figure supplement 1, Beck et al., 2004), further confirming that the behaviour we observe is indeed OKR. Figure 2 with 2 supplements see all Download asset Open asset OKR gain is inferred from a piece-wise fit to the slow phase of tracked eye movements. (a) We present a single pattern of horizontally moving bars to evoke OKR and crop it by superimposing a permanently dark area of arbitrary size and shape (left). Its velocity follows a sinusoidal time course, repeating every 10 s for a total of 100 s for each stimulus phase (right). (b) OKR gain is the amplitude of eye movement (green trace) relative to the amplitude of the sinusoidal stimulus (grey trace). The OKR gain is often well below 1, e.g. for high stimulus velocities as used here (up to 12.5°/s). (c) Even small stimuli are sufficient to elicit reliable OKR, although gains are low if stimuli appear in a disfavoured part of the visual field. Shown here are responses to a whole-field stimulus (top) and to a disk-shaped stimulus in the extreme upper rear (bottom, Figure 2—figure supplement 2). To quantify position tuning, we cropped the presented gratings (Figure 2a) to a disk-shaped area of constant size, centred on one of 38 nearly equidistant parts of the visual field (Figure 3a, Supplementary File 1B, Video 1, Figure 3—video 1). The distribution of positions was symmetric between the left and right, upper and lower, as well as front and rear hemispheres, with some stimuli falling right on the edge between two hemispheres. As permanent asymmetries in a stimulus arena or in its surroundings could affect OKR gain, we repeated our experiments in a second group of larvae after rotating the arena by 180 degrees (Figure 4a–b), then matched the resulting pairs of OKR gains during data analysis (see Materials and methods, Figure 4e–f). Any remaining asymmetries in the OKR distributions should result from biological lateralisation, if indeed there exists such consistent lateralisation across individuals. Figure 3 with 6 supplements see all Download asset Open asset OKR gain depends on stimulus location. (a) The stimulus is cropped to a disk-shaped area 40 degrees in diameter, centred on one of 38 nearly equidistant locations (Supplementary file 1B) across the entire visual field (left), to yield 38 individual stimuli (right). (b–d) Dots reveal the location of stimulus centres D1-D38. Their colour indicates the average OKR gain across individuals and trials, corrected for external asymmetries. Surface colour of the sphere displays the discretely sampled OKR data filtered with a von Mises-Fisher kernel, in a logarithmic colour scale. Top row: OKR gain of the left eye (b), right eye (d), and the merged data including only direct stimulation of either eye (c), shown from an oblique, rostrodorsal angle. Bottom row: same, but shown directly from the front. OKR gain is significantly higher for lateral stimulus locations and lower across the rest of the visual field. The spatial distribution of OKR gains is well explained by the bimodal sum of two von-Mises Fisher distributions. (e) Mercator projections of OKR gain data shown in panels (b–d). White and grey outlines indicate the area covered by each stimulus indicate average gain for stimuli centred on this location. eye position during and on panels indicate stimulation via that stimuli not directly to either the left or right Data from fish for the and fish for the rotated Figure data 1 data and elements of Figure Download Figure data 2 data and elements of Figure Download Figure with 3 supplements see all Download asset Open asset The OKR is upper of fish (a) arena (b) can be 180 degrees front and upper and lower LED positions are The bulb from the of the left and right, upper and lower LEDs are (c) Upside-down embedding (d) with optical including Results in where positive to positions, for the four setups shown in As in Figure colour indicates the discretely sampled OKR data filtered with a von Mises-Fisher and follows a logarithmic colour scale. with presentation of a less distributed of stimuli, cropped to of 64 degrees of the 40 degrees used in (g) exhibit a for stimuli below the that positions to their body (h) as in To for asymmetries such as arena we combined the data underlying (e) and (f) to Figure (see Materials and methods). Data from (e) (f) (g) (h) 10 fish. 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