Quantifying the Ontogeny of Optokinetic and...

Quantifying the Ontogeny of Optokinetic and Vestibuloocular Behaviors inZebrafish, Medaka, and Goldfish

James C. Beck,1 Edwin Gilland,1 David W. Tank,2 and Robert Baker1

1Department of Physiology and Neuroscience, New York University School of Medicine, New York, New York 10016; and 2Departments ofPhysics and Molecular Biology, Princeton University, Princeton, New Jersey 08544

Submitted 29 March 2004; accepted in final form 20 July 2004

Beck, James C., Edwin Gilland, David W. Tank, and RobertBaker. Quantifying the ontogeny of optokinetic and vestibuloocularbehaviors in zebrafish, medaka, and goldfish. J Neurophysiol 92:3546–3561, 2004. First published July 21, 2004; doi:10.1152/jn.00311.2004. We quantitatively studied the ontogeny of oculomotorbehavior in larval fish as a foundation for studies linking oculomotorstructure and function with genetics. Horizontal optokinetic and ves-tibuloocular reflexes (OKR and VOR, respectively) were measured inthree different species (goldfish, zebrafish, and medaka) during thefirst month after hatching. For all sizes of medaka, and most zebrafish,Bode plots of OKR (0.065–3.0 Hz, �10°/s) revealed that eye velocityclosely followed stimulus velocity (gain � 0.8) at low frequency butdropped sharply above 1 Hz (gain � 0.3 at 3 Hz). Goldfish showedincreased gain proportional to size across frequencies. Linearity test-ing with steps and sinusoids showed excellent visual performance(gain � 0.8) in medaka almost from hatching; but zebrafish andgoldfish exhibited progressive improvement, with only the largestequaling medaka performance. Monocular visual stimulation in ze-brafish and goldfish produced gains of 0.5 versus �0.1 for the eyeviewing a moving versus stationary stimulus pattern but 0.25 versus�0.1 in medaka. Angular VOR appeared much later than OKR,initially at only high accelerations (�200°/s at 0.5 Hz), first in medakafollowed by larger (8.11 mm) zebrafish; but it was virtually nonex-istent in goldfish. Velocity storage was not observed except for an eyevelocity build-up in the largest medaka. In summary, a robust OKRwas achieved shortly after hatching in all three species. In contrast,larval fish seem to be unique among vertebrates tested in their lack ofsignificant angular VOR at stages where active movement is requiredfor feeding and survival.

I N T R O D U C T I O N

The vertebrate oculomotor system has aided survival bymaintaining visual acuity during self and visual world motion(Land 1999; Walls 1962) in nearly the same basic form forover 450 million years of evolution (Baker and Gilland 1996).Since the oculomotor system is so universal, lower vertebratesystems are now utilized to link oculomotor behavior with itsgenetic underpinnings. One animal in particular, the zebrafish,Danio rerio, is an established developmental vertebrate model(Fishman 2001), undergoing genome sequencing (Vogel2000). Initial work in zebrafish utilized oculomotor behaviorssuch as the optokinetic response (OKR) to screen for defects inthe retina (Brockerhoff et al. 1995, 1997) as well as earlycomponents of the visual pathway (for review see Baier 2000;Neuhauss 2003; Rick et al. 2000; Roeser and Baier 2003).

However, the oculomotor system has been largely a tool andnot the primary focus of these investigations.

Normally, the visual and vestibular systems work inconcert to produce reflexive compensatory eye movements.In the laboratory, however, these sensory motor systems canbe studied individually (Robinson 1968). The visual sur-round (e.g., striped drum) can be rotated en bloc to producean OKR as the visual system strives to minimize retinal slip(Collewijn 1991). In contrast, the vestibular system lacksany inherent means of feedback in the absence of vision(dark), thus operating in an open-loop mode. Vestibuloocu-lar reflexes (VOR) are evoked by stimulation of two sepa-rate, but complementary, sensory components: the otolithsdetecting linear (L) acceleration and the semicircular canalsdetecting angular (A) acceleration (Angelaki et al. 1995;Hess and Angelaki 1993). These two vestibular reflexes canalso be examined separately. Tilting the head to a fixedangle produces a static change in eye position, while adynamic LVOR is generated by translating the head linearly(Paige 2002). Rotating the animal with the head centered inthe axis of rotation stimulates the semi-circular canals whileminimally affecting the otolith organs, thereby creating aneye movement entirely related to the AVOR (Wilson andMelvill Jones 1979).

To date, only two groups have sought to address the devel-opment of oculomotor function in larval zebrafish. Moorman etal. (1999, 2002; Riley and Moorman 2000) focused on thedevelopment and function of the static LVOR in zebrafish at 5days postfertilization (dpf). Easter and Nicola (1996, 1997)examined the onset and development of optokinetic and angu-lar vestibular behaviors in larval zebrafish aged 2–4 dpf. Thefirst reflexive eye movements appeared at 3 dpf, as slow phaseresponses to a drum rotating constantly at 2.4°/s. Fast phaseresets appeared shortly thereafter and OKR gain (ratio of eye tostimulus velocity) was noted to reach 0.9 by 4 dpf. Horizontalangular VOR, measured with a presumptive ganzfeld (a fea-tureless visual surround), was reported just after the onset ofOKR. Spontaneous saccades appeared prior to 4 dpf andreached adult velocities by 5 dpf. Easter and Nicola (1997)concluded that early larval spontaneous and reflexive optoki-netic behaviors approached adult levels of performance withvestibuloocular behaviors following nearly the same ontoge-netic time course.

Zebrafish represent just 1 of 23,000 extant teleost species(Eschmeyer 1998); thus to ensure that they are representative

Address for reprint requests and other correspondence: J. C. Beck, Dept. ofPhysiology and Neuroscience, MSB-442, NYU School of Medicine, 550 FirstAve, New York, NY 10016 (E-mail: [email protected]).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

J Neurophysiol 92: 3546–3561, 2004.First published July 21, 2004; doi:10.1152/jn.00311.2004.

3546 0022-3077/04 $5.00 Copyright © 2004 The American Physiological Society www.jn.org

examples of vertebrate oculomotor development as a whole aswell as to provide a more complete understanding of oculo-motor function in general, two additional fish species wereselected: goldfish (Carassius auratus) and medaka (Oryziaslatipes). Like zebrafish, goldfish are cyprinids and the twoshare many traits unique to that group. Adult goldfish areextensively used as a neurophysiological structure/functionmodel. OKR (Easter 1972; Keng and Anastasio 1997; Marshand Baker 1997) and VOR (Pastor et al. 1992; Schairer andBennett 1986a,b) have been shown to exhibit robust perfor-mance and adaptation. Purkinje cell activity has been recordedduring normal and learned oculomotor behaviors (Marsh 1998;Pastor et al. 1997), showing roles for cerebellar pathwaysduring the learning, acquisition, and memory phases of oculo-motor plasticity (McElligott et al. 1998; Pastor et al. 1994a).Nearly all key oculomotor related subnuclei have been ana-tomically and physiologically identified, establishing func-tional contributions to short-term memory of eye position andeye velocity (Aksay et al. 2001; Beck et al. 2001; Graf et al.1997; Pastor et al. 1991, 1994b; Suwa et al. 1999). Theoculomotor behavior of the adult goldfish thus represents adevelopmental endpoint by which to compare the progress oflarval oculomotor development.

Unlike goldfish, medaka are distantly related to zebrafish.Members of the beloniforms, a group more derived thancyprinids, medaka are separated from zebrafish by over 110million years of evolution (Nelson 1994). Medaka serves as amodel system for sex determination, environmental toxicity, aswell as developmental studies and is the subject of a currentgenome sequencing project (Ishikawa 2000; Wittbrodt et al.2002). From an early age, medaka exhibit superb visual acuityand optomotor tracking capabilities (Carvalho et al. 2002) andhence provide another attractive candidate for genetic investi-gations of oculomotor behavior.

A general outline of the ontogeny of oculomotor behaviorshas been provided for larval zebrafish (Easter and Nicola1997); however, the examination was limited to only the firstfew days after hatching. In addition, that study lacked thequantification needed to effectively search for subtle differ-ences in mutations that would permit genetic dissection ofoculomotor functions. Here, a quantitative and ontogeneticassessment of the oculomotor system is presented in wild-typezebrafish, goldfish, and medaka from 5–35 dpf.

Part of this work has appeared elsewhere in abstract form(Gilland et al. 2002).

M E T H O D S

Animals (Supplemental Movie 11)

Animals tested were wild-type zebrafish (D. rerio), goldfish (C.auratus), and medaka (O. latipes). Adult goldfish and fertilized eggswere obtained from Hunting Creek Fisheries (Thurmont, MD); ze-brafish and medaka adults were obtained from Aquatic ResearchOrganisms (Hampton, NH) and larvae spawned in the laboratory.Adult goldfish 10–13 cm in length (tip to peduncle) were maintainedand tested at 18°C. Larval goldfish were reared at room temperature(22°C) in small beakers and transferred to small aquaria at �15 dpf.Zebrafish and medaka were raised in an incubator maintained at 28°C.After 10–15 dpf they were transferred to an Aquatic Habitats (Ap-

opka, FL) recirculating system maintained at 28°C. Larval goldfishwere tested at 22°C and zebrafish and medaka at 28°C. Young larvaewere initially reared with a mixture of paramecia and dried food andwere later fed Artemia nauplii and dried food. Animals tested werereared from at least two separate spawns and were exposed to a 16:8h light-dark cycle. Animals were used in accordance with the Guidefor the Care and Use of Laboratory Animals (1996) using protocolsapproved by the NYU School of Medicine Institutional Animal Careand Use Committee.

Adult animals were restrained with an acrylic head holder aspreviously described (Aksay et al. 2000; Marsh and Baker 1997;Pastor et al. 1992). Larval animals, embedded in a drop of low meltingtemperature agarose (2.0%; Sigma Type VII-A), were held horizon-tally (Fig. 1C) in a 19-mm-diam transparent glass specimen chambercontaining 2.5 ml of water (Fig. 1, A and B; also see Movie 1). Thefloor and roof of the specimen chamber were made of round coverslips; the roof was held in place by surface tension and could be leftajar to permit oxygen exchange. The head region, rostral to the swimbladder, was freed of agarose with a scalpel, permitting unrestrictedmovement of the eyes and normal respiration (Fig. 1C, dashed line;Movie 1). Larger animals tended to aspirate the molten agarose thatcould obstruct the pharynx once solidified. This was mitigated bycovering the head with a 6% methylcellulose solution just prior toenveloping with agarose. In addition, large animals could also extri-cate themselves from the agarose once their head was freed; this wasreduced by flexing the tail into a “J” configuration before the agarosesolidified. Alternatively, an electrolytically sharpened tungsten orplatinum 10-�m wire, coated with a 10% benzocaine/ethanol solution,inserted perpendicular to the body through the epithelial ridge in thecaudal tail was sufficient to prevent escape. The solidified agaroseblock was affixed with several 100-�m minutien pins to a 1-mm-thickclear disk (Sylgard 184, Dow Corning, Midland, MI) that snugly fitwithin the bottom of the specimen holder. Animals removed from theagarose swam normally and could be reared to adult stages.

Vestibular and visual stimulation

The vertical axis turntable comprised an aluminum base, housing abelt-drive motor assembly, and an aluminum top connected to the basevia a hollow, stainless steel shaft (Fig. 1A). The surface of the tablecontained 1) a compact, three-axis micromanipulator with a clear,acrylic mounting stage for the specimen holder, allowing the head tobe centered on the table and to focus the animal for the camera; 2) amotorized optokinetic drum; and 3) a CCD camera centered on thetable’s axis. The table motor operated with a servo-controller (modelLDH-S1, Western Servo Design, Hayward, CA) that utilized bothvelocity and position feedback signals.

A small drum centered on the table top provided visual stimuli toinduce optokinetic reflexes (Fig. 1B). Clear acrylic drums of differentsizes, 3.75 and 8 cm, nested concentrically around the specimenholder provided rotating patterned, stationary patterned, or ganzfeld(featureless visual surround) stimuli. For both monocular and binoc-ular testing, moving stimuli on the inner drum and stationary patternedstimuli consisted of alternating black and white stripes (stripe fre-quency 15.5°). The rotating drum was illuminated from below,through the table shaft, by visible light LEDs (Fig. 1B, inset). Thedrum was servo-controlled (model LDH-A1, Western Servo) inde-pendently from the table and also employed both position and velocityfeedback. Stimulus waveforms controlling table and drum motionwere created with two Agilent (Palo Alto, CA) 33120A phase-linked,function generators.

The table and drum operated within a removable, light-tightwooden enclosure with a black felt curtain on the front to facilitateaccess to the specimen. As needed, the enclosure was maintained at28°C with a YSI temperature controller (Yellow Springs, OH) and ascrew base radiant heater (McMaster-Carr, Dayton, NJ).

1 The Supplementary Material for this article (10 movies) is available onlineat http://jn.physiology.org/cgi/content/full/00311.2004/DC1.

3547QUANTIFYING ONTOGENY OF OKR AND VOR

J Neurophysiol • VOL 92 • DECEMBER 2004 • www.jn.org

Oculomotor measurements (Supplemental Movie 2)

Methods for recording eye movements from adult goldfish with ascleral search coil have been extensively described (Aksay et al. 2000;Marsh and Baker 1997; Pastor et al. 1992). Larval eye positions weremeasured and recorded using a real-time video system designed fortracking the horizontal eye movements of larval animals (Beck et al.2004). The specimen holder containing the animals was placed intothe mounting stage and illuminated from below with an infrared LED(Fig. 1C). An initial image was captured (Fig. 1C1, Movie 2) with astandard, infrared sensitive CCD camera attached to a video framegrabber under the control of custom software written in NationalInstruments’ (Austin, TX) LabVIEW programming environment. Aregion of interest (ROI) was drawn around the animal (Fig. 1C2,

Movie 2), the eyes (Fig. 1C3, Movie 2), and a fixed point on the body(Fig. 1C4, Movie 2).

Once the real-time video processing began, the two ROIs selectedaround the eyes and along the body axis were binary thresholded andinverted, producing white eyes (Fig. 1D5, Movie 2) and body refer-ence (Fig. 1D6, Movie 2). The center of mass of the body referencewas then calculated (Fig. 1D7, Movie 2). To select only the eyeswithin the thresholded ROI (Fig. 1D5, Movie 2) and to reject pig-ments (melanophores) in the head around the eyes, the size of eachobject in the eye ROI was computed. The two largest objects in theeye ROI (Fig. 1C3, Movie 2) were the eyes, and the center of mass ofeach was determined (Fig. 1D8, Movie 2). The center of each eyeserved as the starting point for a pixel seeding operation (“magic

FIG. 1. Measuring larval eye movements. View of vestibular turntable (A) and both the inner and outer optokinetic drums (B),depicting potential visual stimuli arrangements. Lying directly below the drum are the visible light and infrared LEDs (B, inset).Also see Supplemental Movie 1. C: to convert 60-Hz video into eye position records, an initial still image was captured (1) andregions of interest (ROIs) drawn around: the entire animal (2, dark border), the eyes (3, black rectangle), and a fixed point alongthe body axis (4, white rectangle). D: during real-time acquisition of video images, the ROIs around the eyes (5) and body (6) werethresholded and inverted allowing determination of the body (7) and eye (8) centers. E: similar pixel values from the center of theeye were filled (9) and the body axis (11) subtracted from the major axis of each eye (10) to determine absolute eye position (12).Dashed line in C is the anterior margin of the agarose block. Also see Supplemental Movie 3.

3548 J. C. BECK, E. GILLAND, D. W. TANK, AND R. BAKER


wand”) that smoothly filled in the entire shape of each eye byselecting neighboring pixels that were of similar intensity (Fig. 1E9,Movie 2). The equivalent ellipse major and minor axes of each filledeye (Fig. 1E10, Movie 2), and the body axis was determined (Fig.1E11, Movie 2). The body axis connected the midpoint between theeyes to the center of the thresholded body reference (Fig. 1D7, Movie2). Eye positions (Fig. 1E12, Movie 2) were calculated as the angle ofthe major axis of each eye (Fig. 1E10, Movie 2) relative to the bodyaxis (Fig. 1E11, Movie 2). Left and right directions were establishedby evaluating the vector cross product of the eye and body axis. Eyepositions, computed with an accuracy �0.1°, were converted on-lineat a rate of 60 Hz to voltages (16 bit) using D/A converters on aNational Instruments computer card (PCI-6052E).

Data acquisition and analysis

Eye position with a 60-Hz temporal resolution plus drum and tableposition, including the visible light LED voltage (for timing of lightON-OFF) were digitized using Axoscope and a Digidata 1200B (AxonInstruments; Union City, CA) at 200 Hz. Data processing was accom-plished using custom routines in MATLAB (MathWorks, Natick,MA) as previously described (Aksay et al. 2000; Major et al. 2004;Mensh et al. 2004). Briefly, eye positions were filtered and digitallydifferentiated to generate eye velocities. Fast phases in eye velocitywere interactively removed by differentiating eye velocity to acceler-ation and setting an acceleration threshold for each behavioral trialrecorded. Acceleration peaks above a manually set threshold, corre-sponding to individual fast phases, as well as 0.15 s before and0.2–0.4 s after each peak were removed by computer. Blinks (Pastoret al. 1991), previously called stretches (Easter 1971), and irregulareye movements were manually removed and excluded from analysis.For video measurements, timing differences due to processing delays(38 ms) were removed between eye position records and other traces.

The amplitude and phase of eye velocity in response to sinusoidalrotation of the drum and table were quantified in MATLAB by leastsquares fitting of eye velocity, over several stimulus cycles, to asinusoidal prototype, defining a single sinusoidal waveform that bestfit the data. Fits were not considered significant if r2 � 0.5. Eyevelocity gain was calculated as the ratio of the peak-to-peak amplitudeof the sinusoid fit to eye velocity to the peak-to-peak amplitude of thestimulus velocity. The phase difference was taken as the difference indegrees between the peak of the stimulus and the corresponding peakof eye velocity: positive differences represent phase leads. For stepstimuli, eye velocities were calculated as the mean velocity for thefirst one-half (early gain/build-up) and second one-half (sustainedgain) of several 0.1-Hz bidirectional step cycles, excluding 0.1 s oneither side of the step transition (Collewijn 1969; Lisberger et al.1981). Binocular data analysis was performed by averaging results forboth eyes. For monocular analysis, results for the moving eye versusthe stationary eye were averaged separately with respect to the visual

stimuli presented to each. Saccade analysis used the slope of a linearregression (least absolute deviation) fit to the maximum saccadic eyevelocity (unfiltered) versus the saccadic position amplitude for oneeye. Leftward saccades were positive and rightward saccades werenegative. For one eye, this slope calculation was functionally equiv-alent to the use of absolute values of velocity and amplitude andregressing through the origin (�1% difference).

Statistical tests were performed using Igor Pro 4.0 (Wavemetrics,Lake Oswego, OR). ANOVA was used to examine differences inperformance from sinusoidal frequency and linearity testing as well asduring velocity steps. Multiple group and individual comparisonswere made, post hoc, among all size classes for each treatment, i.e.,specific stimulus frequency/amplitude, with the Student-Newman-Keuls test (Norman and Streiner 2000; Zar 1996). A Student’s t-testwas used to compare eye velocity gains from initial/build-up andsustained parts of velocity steps (1st and 2nd halves of a step).Significance was determined for P � 0.05. Unless otherwise stated,results are reported as �SE.

Quantitative measurements of oculomotor performance were cate-gorized according to fish length. Recent work has concluded that sizenot age is the best metric of fish maturation (Fuiman et al. 1998; Higgset al. 2002). Since many variables may affect fish growth (tempera-ture, light cycle, and/or husbandry; Davis et al. 2002; Kamler 2002),reporting fish length permits a more effective comparison of resultsbetween experiments. To measure animal size, photographs weretaken through a dissecting scope with a 3.34-megapixel digital camera(Nikon 990). Total animal length was measured in pixels against acalibrated standard using the public domain ImageJ program (Na-tional Institutes of Health). A size distribution histogram was used togroup animals based on clusters of similarly sized individuals (seeTable 1).

R E S U L T S

General considerations

In total, the oculomotor performance of over 300 zebrafish,goldfish, and medaka larvae, from shortly after hatching (5, 7,and 10 dpf, respectively) to the juvenile-larval transition (�35dpf), were examined during preliminary and final experiments.Larvae utilized for testing were chosen from age-specificcommunity tanks and were 1) healthy in appearance (e.g.,inflated swim bladder, active feeding, robust evasive response)and 2) of average size relative to peers. Some of the larvaeprepared for testing (20%) were excluded from analysis whenthey either failed to perform fast phases during an initiallow-frequency (0.065 Hz) optokinetic stimulus (�10%) orexhibited a sudden drop in performance during testing, alsousually an inability to make fast phases (�10%). Based on

TABLE 1. Class number and larval body length for OKR, constant velocity saccade, performance, and VOR plots

Size/ageclass

Zebrafish Goldfish Medaka

Meanlength,

mmRange,

mm

MeanAge,dpf n

Meanlength,

mmRange,

mm

MeanAge,dpf n

Meanlength,

mmRange,

mmMean Age,

dpf n

1 3.91 � 0.02 3.81–4.01 6.4 � 1.0 11 5.95 � 0.05 5.76–6.08 9.83 � 1.2 6 5.00 � 0.06 4.57–5.42 10.7 � 0.45 152 4.22 � 0.03 4.10–4.42 12.7 � 1.7 14–15 6.25 � 0.03 6.17–6.36 13.3 � 2.5 7 6.21 � 0.06 5.88–6.46 17.2 � 0.88 93 4.73 � 0.03 4.53–4.93 15.9 � 1.5 16 6.56 � 0.03 6.41–6.75 16.3 � 1.9 14–16 7.65 � 0.10 6.85–8.15 22.5 � 1.0 164 5.25 � 0.05 5.06–5.47 21.7 � 1.7 6–7 7.08 � 0.05 6.91–7.26 30.1 � 2.7 8 8.69 � 0.08 8.45–8.88 31.7 � 1.7 65 5.98 � 0.06 5.72–6.13 24.2 � 1.5 6 7.64 � 0.06 7.36–7.97 31.7 � 2.7 11 9.29 � 0.08 9.09–9.46 31.3 � 1.3 46 6.86 � 0.08 6.42–7.35 26.4 � 0.7 11 8.78 � 0.24 8.29–9.34 23.8 � 3.6 47 8.11 � 0.29 7.62–8.94 33.8 � 1.3 4

Values are means � SE. OKR, optokinetic reflexes; VOR, vestibuloocular reflexes.



preliminary experiments and comparison with otherwise iden-tical age peers, it was clear that these animals were not merelybelow average in performance but were acutely compromised,possibly from the stress of agarose embedding. From thisremaining pool (80%), 71 zebrafish, 53 goldfish, and 51medaka were selected for analysis (Table 1).

Optokinetic velocity steps

Velocity steps are a means of assessing oculomotor perfor-mance by testing the linearity of the optokinetic system withstimuli of increasing amplitude (10–50°/s). Figure 2 showsrepresentative responses of zebrafish (goldfish were similar)and medaka to bi-directional drum velocity steps at 0.1 Hz.Previous studies of the vertebrate visuomotor system separatedthe optokinetic reflex into early components (1st 200 ms) andindirect (delayed) components for analysis (Cohen et al. 1977;Collewijn 1969). The temporal resolution of the video trackingsystem (17 ms) limited accurate quantification of the latencyand acceleration during the early component. Therefore anal-ysis focused on eye velocity during the longer, delayed com-ponent (Collewijn 1969; Lisberger et al. 1981). The delayedcomponent has been divided into two parts: 1) an early rise ineye velocity that gradually increases, termed “build-up”, thatplateaus during the course of a step into 2) a sustained level ofeye velocity (Cohen et al. 1977). The gain (ratio of eye velocityto stimulus velocity) of the initial rise and build-up in eyevelocity in the first 2.5 s of the step was calculated andcompared with the gain during the sustained component of eyevelocity, here, the last 2.5 s of the step (see Fig. 2F).

At low step velocity (10°/s, Figs. 2, A and B, and 3), theinitial and sustained gains of both small and large animals werequite similar. However, as step velocity increased, two trendswere clear among the cyprinids: 1) initial gains were greaterthan sustained gains and 2) overall performance increased withsize (Figs. 2, C and D, and 3, A–E). OKR step performanceimproved for zebrafish �5.98 mm and for goldfish �7.08 mm.For these larger animals, initial gains were significantly greaterthan the smaller sized animals (20–50°/s; ANOVA, P � 0.05),and initial and sustained gains were nearly of equal amplitude(paired t-test; Fig. 3, D and E, *). In addition, there appeared tobe a difference in fast phase resets between small and largecyprinids. At 10°/s (Fig. 2, A and B), small animals madefewer, larger amplitude (position change) resets than largeranimals, although both exhibited comparable eye velocitygains. With increasing velocity (Figs. 2, C and D, and 3, A–E),the fast phase amplitude in smaller zebrafish and goldfish couldnot match that of larger animals and, sometimes, resets failedto occur (Fig. 2C, *). Even though larger animals made morefast phases, eye velocity could not always be maintainedthroughout the step (Fig. 2D, arrows).

Medaka, in contrast, showed almost no disparity betweeninitial and sustained gains or increased performance with size.

E

FIG. 2. Optokinetic velocity steps. Optokinetic reflex (OKR) performancein small (A) and large (B) zebrafish visually stimulated with drum velocitysteps of �10°/s at 0.1 Hz. Vertical dashed lines in position traces show that thenumber of resetting fast phases was greater for the larger animal. C: decreasedOKR eye velocity gain with increased step amplitude (40°/s, same animal asA). *Resets sometimes failed to occur. D: larger zebrafish were not able tomaintain eye velocity during the course of a 50°/s step (arrows), but both small(E) and large (F) medaka closely followed drum velocity. Arrows show thatsustained eye velocity in the latter was greater toward the end of the step thanduring early/build-up eye velocity. In each record, the top traces show left(black) and right (gray) eye position, and the bottom traces show left (black)and right (gray) eye velocity and stimulus velocity (dashed line). Verticaldashed lines in the eye position records are fast phases and have been removedfrom the velocity traces. Velocity and position scale bars are to the left; 0°represents eye position perpendicular to the body axis. Leftward eye move-ments are up, rightward down. Gain values are average of entire step; timescale in F applies to all panels.



Even with large amplitude steps (�50°/s), medaka of all sizesmaintained an eye velocity that closely matched drum velocitythroughout the step, and all animals exhibited a high-frequencyof fast resetting phases (Figs. 2, E and F, and 3C). Like somezebrafish, larger medaka showed a build-up in eye velocityduring the step with the sustained gain greater than the initialgain (Figs. 2F and 3C, arrows), a signature of an activevelocity storage mechanism (Cohen et al. 1977; Collewijn1991).

Optokinetic after-nystagmus

The observation of increased eye velocity during the courseof the velocity steps, i.e., sustained � initial gain, suggestedthe development of a central neural mechanism for velocitystorage (Cohen et al. 1977). A more direct indicator of velocitystorage is the persistence of eye velocity after the cessation ofstimulation in the form of optokinetic after-nystagmus(OKAN). During OKAN in adult goldfish, eye velocity andfast phases continue after the light is extinguished with velocitydecaying exponentially to 0°/s with a time constant (�) �75 s

(Marsh and Baker 1997), with � the measure of velocitystorage (Cohen et al. 1977). To measure � in larval fish, thedrum, operating at a constant velocity (20°/s) was illuminatedfor 20–30 s, and then the light was extinguished. There was noobservable OKAN in any larval animal tested, and � wasconsistently �0.5 s for all three species (Fig. 4, data notshown). Even among the largest animals that showed increasedsustained eye velocity with bi-directional steps (e.g., Fig. 2F),only slight increases in � were observed when quantified withexponential fitting (Fig. 4C).

Saccade metrics

The apparent difference in both the frequency and amplitudeof saccades between small and large animals, notably inzebrafish and goldfish, prompted a closer assessment of sac-cade performance (Fig. 5). The frequency of spontaneoussaccades was examined under both light and dark conditions.Spontaneous activity in the light was higher in zebrafish than ingoldfish or medaka and increased almost fourfold with matu-rity (Fig. 5A, filled points). Goldfish saccade frequency in the

FIG. 3. Summary of optokinetic velocity step performance. OKR performance in response to visual step stimuli 0.1 Hz,�10–50°/s is shown for zebrafish (A and D), goldfish (B and E), and medaka (C and F). Eye velocity gain was divided into 2 parts:early/build-up (A–C) and sustained (D–F, also see Fig. 2F). *Gain of sustained eye velocity was often significantly less than duringthe early/build-up phase. Legend shows color code for size, symbols for early/build-up (solid) and sustained gain (empty) alongwith animal number for each size class. Table 1 details animal length and age for each size class. Traces for each size class havebeen horizontally offset in this and the following figures to facilitate viewing. Error bars are �SE.



light remained relatively constant and was generally lower thanmedaka until a large increase at size class 6. Saccade frequencyin the dark was slightly lower than in the light for bothcyprinids at all sizes (data not shown). Initially, almost nosaccades were made by larval medaka in the dark; however, bythe end of the study period, the largest medaka (class 5) weremaking saccades only slightly less frequently (0.12 � 0.02 Hz)than in the light. The frequency of resetting fast phases, drivenwith an optokinetic stimulus (30°/s velocity step), roughlyquadrupled over spontaneous levels in zebrafish and increasedover eightfold in goldfish and medaka (Fig. 5A, open symbols).

The increase in resetting frequency with size largely paralleledthat seen for spontaneous saccades. Larval goldfish did notmake as many spontaneous saccades as adults (Fig. 5A, lonesquares); but when optokinetically stimulated, the largest lar-vae made a comparable, and sometimes greater, number ofresets as adults.

Although frequency is important, fast phase resets need to beof sufficient amplitude and velocity to reposition the eyes in theorbit to maintain slow phase eye velocity and minimize loss invision (Carpenter 1991; Collewijn 1970; Walls 1962). Becausesaccade velocity is generally proportional to the change in eyeposition (saccade amplitude), the slope of maximum velocityversus amplitude (Collewijn 1970; Easter 1975; Fuchs 1967;Mensh et al. 2004; Zuber et al. 1965) provides another usefulmetric for saccade performance. Saccade velocity-amplitude(VA) slopes were calculated for both spontaneous and OKRbehaviors (Fig. 5B). In zebrafish and goldfish, saccade VAslopes during OKR were not significantly higher than duringspontaneous saccades until the largest sizes. In contrast, allmedaka had steeper VA slopes during optokinetic resets thanduring spontaneous saccades. Increases in saccadic VA slopewith size were modest for zebrafish and medaka but muchgreater for goldfish (Fig. 5B). In particular, the sigmoid-shapedsize-VA relationship for larval goldfish almost doubled by sizeclass 3, preceding improvements in velocity step gain, andincreased again at class 6. Adult goldfish spontaneous saccadicperformance (Easter 1975; Mensh et al. 2004) was similar tothat of the largest larval goldfish but exhibited a steepersaccade VA slope during optokinetic resets (Fig. 5B).

Optokinetic sinusoids (Supplemental Movie 3)

Smaller larval animals were often unable to maintain aconstant, peak eye velocity during the 5-s period of the drumvelocity step (Figs. 2C and 3, A–E). Consequently, sinusoids ofincreasing amplitude (10–50°/s at 0.125 Hz; Fig. 6) werechosen as another means of assessing the linearity of theoptokinetic system because stimulus velocity varies with timeand maximum velocities are reached for only brief periods.Behavioral performance was characterized by both eye veloc-ity gain as well as the phase of eye velocity relative to thestimulus (Fernandez and Goldberg 1971; Melvill Jones andMilsum 1971; Robinson 1981). As seen with velocity steps, allthree species could follow low-amplitude (10°/s) sinusoidswith high eye velocity gain and minimal phase shifts (Fig. 7).With increasing stimulus amplitude, performance for bothcyprinids dropped (decreased gain, leads in phase) but laterimproved with size (Figs. 6, A–C and 7, A and B; Movie 3). In

FIG. 4. Eye velocity storage. Optokinetic after-nystagmus (OKAN) and velocity storage were absent in goldfish (A), zebrafish(B), and medaka (C, same animal as Fig. 2F). Exponential fitting of the decrease in eye velocity (thick line) revealed extremelyshort decay time constants (�).

FIG. 5. Saccade frequency and performance. A: saccade frequency mea-sured during spontaneous eye movements in the light and during a 30°/soptokinetic velocity step for zebrafish, larval and adult goldfish, and medaka(see B, inset). B: slope of peak saccade velocity vs. saccadic amplitude for bothspontaneous saccades in light and during optokinetic resets. Table 1 details sizeclass and animal number; adult goldfish, n � 5.



the smallest cyprinids, the eyes would occasionally lock in anextreme position but did so much less frequently than duringvelocity steps. Overall, sinusoidal linearity improved steadilywith maturity, with statistically significant differences princi-pally between the smallest and largest sized animals. Eyevelocity gain from 30–50°/s and phase leads from 40–50°/s ofsmall zebrafish (classes 1–3) were significantly lower(ANOVA, P � 0.05) compared with the largest size class (Fig.7A). Mid-sized zebrafish (classes 4–6) showed improved lin-earity with size and were only significantly less than the largestzebrafish at 50°/s. Goldfish linearity showed the most changewith size (Fig. 7B). The largest size class performed signifi-cantly better than classes 1–2 at 20 and 30°/s and significantlybetter than classes 1–3 at 40 and 50°/s (ANOVA, P � 0.05).The phase lead of the smallest goldfish was significantlygreater than the others across all stimulus velocities.

In contrast to zebrafish and goldfish, all medaka followed thesinusoidal drum stimulus extremely well with gains �0.8 forall stimulus velocities (Fig. 6, B and D, and 7C). Although thephase lead of the smallest medaka was significantly greaterthan larger medaka above 10°/s (classes 2–5), this phasedifference was minimal compared with that observed for smallzebrafish and goldfish. Overall, medaka was more linear inboth gain and phase than either goldfish or zebrafish at allsizes. As with steps, only the largest cyprinids were able toperform as well as small medaka.

Optokinetic frequency bandwidth (Supplemental Movies 4, 5,and 6)

Bode frequency plots are a useful way to characterize theinput-output relationship of the oculomotor system using sinu-soidal stimuli of varying frequency at a constant velocityamplitude (Robinson 1981), here, 0.065–3.0 Hz and �10°/s. Itwas at this amplitude that larvae of all sizes exhibited nearly

equal performance during sinusoidal linearity testing. Repre-sentative tests showing differences in performance between thesmallest and largest animals as well as between species areshown (cf. Fig. 8, A vs. B vs. C; also see Movie 4, Movie 5).The overall change in frequency bandwidth is summarized inBode plots made of eye velocity gain and phase versus fre-quency for each size class in Fig. 9.

In general, all three species were able to follow low-frequency drum oscillations with high gain within the firstfew days after hatching (Fig. 8, A–C, smallest sizes). Eyevelocity gain and phase were largely steady from 0.065 to1.0 Hz; higher stimulus frequencies yielded decreasing gainwith increasing phase lag. At low stimulus frequencies,zebrafish and medaka exhibited minimal improvement inOKR with increased size while goldfish showed a progres-sive increase. However, at 3 Hz, the highest frequencytested, both gain and phase improved significantly duringzebrafish maturation from the smallest size (Figs. 8G and9A; ANOVA, P � 0.05). Goldfish performance qualitativelyimproved with size, exhibiting significant improvements atmost frequencies (Figs. 8, B, E, and H, and 9B; 4/7;ANOVA, P � 0.05), with post hoc analysis showing thegain in the two smallest size classes to be significantly lessthan the larger groups at 3 Hz (Figs. 8H and 9B). Eyevelocity gain and phase of even the smallest medaka sur-passed those of the largest cyprinids at most frequencies. At3 Hz, phase shifts were similar among the larger animals ofall three species, though eye velocity gain in goldfish wasnearly double that of zebrafish and medaka (Fig. 9). Anotherbehavioral feature that was observed in the digitized recordsand confirmed by visual observation was the appearance ofjerky eye movements among the smaller sizes of all speciesand often in goldfish and medaka (Fig. 8, B and F, insets;Movie 6).

FIG. 6. Sinusoidal linearity testing. OKR performance of small and large zebrafish (A and C) and medaka (B and D) at thehighest stimulus velocity tested, 50°/s at 0.125 Hz (Movie 3). Vertical dashed lines in A show fast resetting phases where eyevelocity decreased in the small zebrafish. Trace markings and calibrations are as in Fig. 2.



Monocularity (Supplemental Movie 7)

During each of the preceding oculomotor tests, all threespecies exhibited extremely conjugate eye movements. Toassess the independence of eye motion, stripes were removedfrom one-half of the rotating optokinetic drum (180°) to revealstripes on the stationary outer drum. As a result, one eye waspresented with a stationary, patterned stimulus and the otherwith a patterned stimulus that rotated sinusoidally at 0.125 Hzand �20°/s (Fig. 10A; Movie 7). The eye following the movingstimulus showed no obvious nasal/temporal asymmetry in eyevelocity while the other eye remained relatively still (Fig. 10A;Movie 7). Saccades were of the same direction but of differingamplitudes. The moving eye in zebrafish and goldfish had ahigher velocity than in medaka (Fig. 10B, solid markers; Movie7). Zebrafish exhibited a significant decrease in monocular gainwith maturity (ANOVA, P � 0.05). An unexplained drop

occurred in goldfish size class 2, which included animals (n �4) from two spawns and three separate recording sessions. Forall species, the gain of the stationary eye was low (�0.1, openmarkers, Fig. 10B). The phase difference was near 0° for themoving eye in cyprinids, but small medaka exhibited a phaselead of 15° that steadily decreased toward 0° with maturation(data not shown). In small zebrafish, the stationary stripes werereplaced with a featureless, gray ganzfeld and the moving drumrotated as before (Fig. 10C, n � 3, 2, 3). The velocity of themoving eye was not significantly different in the smallestzebrafish compared with binocular stimulation but decreasedsignificantly in larger fish (ANOVA, P � 0.05). Eye velocityof the ganzfeld viewing eye remained unchanged.

Angular VOR (Supplemental Movies 8, 9, and 10)

Angular VOR was evoked with horizontal sinusoidal andstep rotation in the dark. The earliest compensatory eye move-ments were observed in about one-half (6/15) of the smallestsized medaka. With a table stimulus of 1 Hz, �120°/s (�680°/s2), medaka eye velocity was very small (gain � 0.19 �0.05and phase lead of 31 �5° not shown). Surprisingly, neither thesmallest sized zebrafish (Movie 8) nor goldfish exhibited anAVOR when tested with sinusoids or steps with accelerations�500°/s2. In comparison, the gain of AVOR in adult goldfishaveraged 0.9 at 0.125 Hz, �16°/s—12.6°/s2 peak acceleration(Pastor et al. 1992; Schairer and Bennett 1986a). Notably, noneof the larval animals �14 dpf exhibited any observable low-frequency AVOR, e.g., at 0.125 Hz, �12.5°/s2. In addition, eyemovements in all but the largest cyprinid larvae were generallyof such low amplitudes as to be imperceptible by visualobservation of the live video image. Although changes in eyeposition could be measured with the eye tracking algorithm(Fig. 11A), least squares fitting of the expected sinusoid to theeye velocity traces mostly yielded unreliable Pearson coeffi-cients (r2 � 0.5). As cyprinids grew in size, AVOR appearedcapriciously, and eye velocity gain was never �0.25 and rarely�0.10 at any frequency and/or amplitude during the studyperiod (max, 1 Hz, �120°/s sinusoids or 0.5 Hz �60°/s steps).The best performing cyprinid was a zebrafish shown in Fig.11B. An intermediate acceleration was chosen for analyzingmaturation of AVOR in larval medaka (table stimulus of 0.5Hz, �60°/s, �190°/s2). A plot of AVOR eye velocity gain andphase versus size showed eye velocity increased from zero inthe smallest animals to a gain and phase in the largest that werecomparable to adult goldfish (Fig. 11, C and D, Movie 9).

The vestibular responses to table velocity steps in the dif-ferent medaka size classes paralleled the sinusoidal responses.During table steps of �40°/s (peak 190°/s2), small- to medium-sized larvae generally showed either small or no transientresponse (10–15°/s amplitude; Fig. 12A). These small stepresponses were generally seen in cases that also exhibited loweye velocity gain evoked by sinusoidal stimuli. High gain eyevelocity responses to head velocity steps were only seen in thelarger-sized medaka. Progression from minimal responses toeye velocity pulses briefly reaching above unity gain are shownfor medaka 7.2–8.8 mm in length (Fig. 12, A–C). For inter-mediate-sized animals, eye velocity gradually increased duringthe first few successive steps of table movement (Fig. 12B,arrows). Even for the largest animals with a strong compensa-tory response (Fig. 12, C and D; Movie 10), eye velocity was

FIG. 7. Summary of sinusoidal linearity testing (10–50°/s at 0.125 Hz).Gain and phase plotted for each velocity tested in all size classes of zebrafish(A), goldfish (B), and medaka (C). Legend shows color code for size, symbolsfor gain (solid) and phase (empty), and animal number for each size class.Table 1 details fish length and age for each size class.



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of short duration relative to the step length. Stimulated with apeak acceleration �600°/s2 (Fig. 12D, same animal as C), eyevelocity quickly returned to 0 in �500 ms, showing the shortvestibular time constant typically observed in this study.

D I S C U S S I O N

In contrast to the results and conclusions of Easter andNicola (1997), quantitative analysis of the eye movements ofdeveloping zebrafish revealed distinct differences in oculomo-tor performance that varied not only with maturity; and, asshown here with goldfish and medaka, these differences alsoextend across species. Notably, optokinetic performance wasrobust from just after hatching both in the cyprinids andmedaka, while AVOR was entirely absent until many days

later. Visual tracking in the two cyprinids showed progressiveimprovement, whereas that of medaka remained relativelyunchanged from the early, robust performance. Angular VOR,absent at first in all fish, matured progressively and appearedfirst in the medaka, followed significantly later in zebrafish andgoldfish.

Visual performance

Bode plots of larval optokinetic bandwidth, performed withthe use of sinusoids, showed that all three species exhibitedrelatively high eye velocity gains with minimal phase shifts atlow frequencies (Fig. 9); however, as frequency increased, gaindecreased and phase lagged. These low-pass filtering charac-teristics were not unexpected as they are common to allvertebrate optokinetic systems (Collewijn 1991). The jerky eyemovements observed in small animals (Fig. 8, B and F, insets)may be a common developmental feature related to the recruit-ment of a limited number of motor units. OKR frequencyperformance increased with maturity, gradually in goldfish(Fig. 9B), more rapidly in zebrafish (Fig. 9A), but almostimmediately in medaka (Fig. 9C). Both visual motion process-ing as well as optical properties of the eye could play a role inperformance bandwidth (Carpenter 1991). However, shortlyafter hatching, both larval medaka and zebrafish exhibit com-parable visual acuity of �5°, which is much less than the 15°stripes used here (Carvalho et al. 2002; Clark 1981). Never-theless, a possibility that was not systematically tested in thisstudy was that maturation of visual motion sensitivity mayproceed at different rates for different target spatial frequen-cies.

The bandwidth in larval animals compared favorably toother afoveate, lateral-eyed animals, such as rabbit (Collewijn1969), rat (Hess et al. 1985), and mouse (van Alphen et al.2001) and was equal to or greater than the adult goldfish (Kengand Anastasio 1997; Marsh and Baker 1997; Schairer andBennett 1986b). A comparison of eye velocity at a singlefrequency (0.125 Hz) showed that larval eye velocity at either10 or 20°/s was almost double that reported for adult goldfishat 16°/s (Marsh and Baker 1997; Schairer and Bennett 1986b:gains of 0.45 and 0.41, respectively). Perhaps, the reason forthe difference is the high-contrast, striped drum used for larvalvisual stimuli. Adult goldfish eye velocity was also higher(gain � 0.6; Keng and Anastasio 1997) when tested with astripe drum than with a planetarium projecting spots of light(Marsh and Baker 1997; Schairer and Bennett 1986b), al-though still less than larval animals. It is therefore possible thisdifference in eye velocity reflects a true age-dependent de-crease in OKR gain performance concomitant with the subse-quent acquisition of AVOR. With both visual and vestibularcomponents of the oculomotor system fully functional in theadult, OKR gain may need not be so high to generate compen-satory eye movements.

Velocity steps and OKAN

Optokinetic steps reveal two well-studied components ofvisual behavior: an early, short latency pathway (presumedpretectum to brain stem) and a delayed, longer time-coursepathway that builds-up eye velocity (presumed to include brainstem-cerebellar pathways, Cohen et al. 1977; Fuchs and Mus-

FIG. 9. Bode plots of optokinetic performance across size classes. OKRgain and phase from 0.065–3.0 Hz, �10°/s (A–C) is shown for zebrafish (Z),goldfish (G), and medaka (M). Legend shows color code for size, symbols forgain (filled) and phase (empty), and animal number for each size class. Table1 details fish length and age for each size class.



tari 1993). Activation of the early pathway is clearly evident inthe records of all larval fish (Fig. 2) and appears as the rapidchange in eye velocity, occurring �200 ms after a change indrum direction. In adult animals, the delayed pathway com-prises the remaining portion of the velocity step and exhibits arelatively slow build-up in eye velocity until a sustainedvelocity is reached (Cohen et al. 1977; Collewijn et al. 1980;Marsh and Baker 1997). Instead of a gradual increase, mostlarval fish showed either no build-up (Fig. 2E) or an actualdecrease in sustained eye velocity (Fig. 2D, arrows). Evidencefor slow build-up in the indirect pathway was only seen in thelargest medaka that exhibited a step response indicative ofvelocity storage (Fig. 2F, arrows).

The presence of a short-term memory of eye velocity (Cohenet al. 1977) after a prolonged velocity step is a direct measureof the velocity storage neural integrator, which is purported toaid spatial orientation during tilt and translation (Fuchs andMustari 1993; Wearne et al. 1999). While velocity storage hasbeen readily observed from adult goldfish (Marsh and Baker1997) to primates (Cohen et al. 1977), evidence of storage inthe form of OKAN was not observed in any larval fish duringthe study period (Fig. 4). Because velocity storage is dependenton the activity of the vestibular nucleus (Cohen et al. 1973,1983; Waespe et al. 1985) as well as the velocity integrator(Cannon and Robinson 1987; Cheron et al. 1986; Pastor et al.1994b), it is possible that one, or both, of these brain stemneural structures are not fully active in larval zebrafish andgoldfish, which lack both velocity storage and an AVOR. Italso might be expected that velocity storage would follow

shortly after the appearance of robust AVOR in larval fish.Since the largest medaka showed the greatest eye velocitybuild-up as well as the most robust VOR, this might indicatethe time when the vestibular nucleus and a velocity integratorare just beginning to function.

Optokinetic linearity and saccades

At low stimulus velocities (10°/s), zebrafish, goldfish, andmedaka of all sizes showed a comparable ability to followvisual stimuli (Figs. 3 and 7). As velocity amplitude increased(20–50°/s), medaka exhibited a linear response in oculomotorperformance with eye velocity gain remaining unchanged. Incontrast, zebrafish and goldfish showed a nonlinear decrease ineye velocity gain with increasing stimulus amplitude. Perfor-mance with higher velocity stimuli improved steadily withincreasing size in the cyprinids, especially with sinusoidalstimulation (Fig. 7, A and B). The improving ability to maintaina high eye velocity might depend on several factors. Since eventhe smallest cyprinids could initially follow high-amplitudestimuli (e.g., Fig. 2C), it is unlikely that the ability to generatehigh velocity is a limiting factor. Decreased vestibular activitycould also play a role as VIIIth nerve lesions affect the abilityto adequately follow step stimuli for several months (Collewijn1976). However, the most parsimonious explanation, sug-gested by the results, is that the frequency and amplitude ofsaccadic resets significantly limits maintenance of high eyevelocity in zebrafish and goldfish, but not medaka.

Spontaneous saccade and optokinetic reset frequency weredirectly correlated with maturity in zebrafish and medaka butless so in goldfish (Fig. 5A). The frequency of optokineticresets was highest in medaka, paralleling its superior linearityperformance. The increases in saccade frequency in zebrafishsize class 5 and goldfish size class 6 corresponded to increasesin gain during velocity steps, but no such change in frequencywas observed in the increase in step gain for goldfish sizeclasses 2–4 (cf. Figs. 3 and 5). Thus, although saccadicfrequency correlates with some aspects of optokinetic matura-tion, it is an unreliable measure of eye velocity (see Fig. 2D)and should not be used as a direct measure of oculomotor

FIG. 10. Monocular optokinetic stimulation (Movie 7). A: monocular OKR for a 5.7-mm, 20 dpf zebrafish. Each one-half of thevisual field was stimulated with a striped drum (dashed line): either stationary (right, gray traces) or rotating sinusoidally at 0.125Hz, �20°/s (left, black traces). B: gain of moving and stationary eye across size classes shown for all species. Animal size classand number as in Table 2. C: gain of zebrafish eye velocity for binocular stimulation (filled squares) as well as monocularstimulation with 1 eye viewing a moving pattern (filled symbols) and the other either a stationary pattern (filled/empty circles) ora ganzfeld (filled/empty triangles).

TABLE 2. Larval class number and n for monocularity testing plot

Size/Age Class Zebrafish Goldfish Medaka

1 10 4 62 12 4 83 14 11 154 3 7 45 6 7 16 97 3



performance (e.g., Rick et al. 2000). The relationship betweensaccade velocity and amplitude (Collewijn 1970; Easter 1975;Fuchs 1967; Mensh et al. 2004; Zuber et al. 1965) yields aslightly different view of the influence saccadic maturation hason optokinetic tracking. The increase in goldfish OKR gain(Figs. 3B and 7B) corresponded with the rise in VA slope atsize classes 3–6, while a correlation in zebrafish was lessevident. Interestingly, only medaka and the largest larval cyp-rinids were able to increase resetting amplitude-velocity inresponse to an optokinetic stimulus (Fig. 5B), which, in part,may have contributed to the improved eye velocity gain and

phase achieved by these animals at high stimulus amplitudes.Overall, goldfish showed different developmental profiles thanzebrafish or medaka for both measures of saccade perfor-mance. This contrasts with the general similarity in maturationof OKR linearity performance in zebrafish and goldfish relativeto medaka (Figs. 3 and 7) and suggests that the performancecontributions of saccade frequency versus saccade VA slopediffer between the three species.

Monocularity

All of the animals tested showed conjugate eye movementsin response to bilateral presentation of visual stimuli. In lateral-eyed animals, the visual fields of the two eyes do not exten-sively overlap; the visual motion perceived by each eye can be

FIG. 12. Medaka angular vestibuloocular reflex in response to table veloc-ity steps. A: velocity steps at 0.5 Hz, �45°/s elicited a transient response in amedium-sized animal. B: in the next size class (4), velocity steps evoked aprogressively increasing (arrows) but brief transient response. High-frequencyoscillations in the eye velocity record are due to respiratory movements. C: ananimal from the largest size class produced a larger gain but eye velocitydecayed to 0°/s. D: at an even lower amplitude velocity stimulus (�20°/s), theanimal exhibited a transient response but lasted �500 ms (Movie 10). Recordsin A–D as in Fig. 2. Head velocity trace (dashed line) is inverted.

FIG. 11. Angular vestibuloocular reflex (AVOR) at 0.5 Hz, �60°/s. A:AVOR was absent in a typical representative cyprinid: 7.8-mm, 30 dpfzebrafish (also see Movie 8). B: best AVOR measured in cyprinids was a largezebrafish (gain � 0.20). C: typical performance for a large medaka with highereye velocity gain (0.51; Movie 9). D: summary of AVOR development at 0.5Hz, �60°/s in medaka. Phase data for the smallest size could not be accuratelyquantified. Records in A–C as in Fig. 2. Head velocity trace (dashed line) isinverted.



combined centrally to generate binocular eye movements.Experiments in mammals (Simpson et al. 1979) and pigeon(Gioanni et al. 1983) have localized one site for this centralneural processing of horizontal visual slip to the accessoryoptic system and nucleus of the optic tract in the pretectum (forreview, see Simpson et al. 1988). Zebrafish and goldfishshowed an ability to almost completely dissociate the motionof the two eyes during monocular stimulation with rotatingstripes and a stationary pattern, while medaka were less adeptat this task. Unlike observations in other developing animals(Braddick and Atkinson 1983; van Hof-van Duin 1978; Wall-man and Velez 1985), a strong temporal-nasal preference toeye velocity gain was not observed. Interestingly, eye move-ments became less decoupled with maturity in zebrafish whentested with either a ganzfeld or stationary pattern. In light ofrecent work on CNS asymmetries in zebrafish (Concha et al.2000; Essner et al. 2000; for review, see Halpern et al. 2003;Liang et al. 2000), monocular eye movements may offer asuitable way to correlate structural changes in neural asymme-try with behavior.

AVOR

Previous investigations of angular VOR in larval fish re-ported a robust compensatory VOR in larval zebrafish as earlyas 4 dpf using low acceleration stimuli (�12°/s2 peak; Easterand Nicola 1997). However, in that investigation, AVOR wasmeasured in the light, and visual feedback was not eliminated.Here, when AVOR of small larvae was tested in the dark, noeye movements were observed at low accelerations. Moreover,a significant AVOR was observed only in larger animals withhigh acceleration canal stimuli (�180°/s2). Medaka were thefirst to consistently produce an AVOR, and they showedprogressive increase in gain with size (Fig. 11D). Zebrafishdeveloped a significant AVOR only in the largest sizes (Fig.11B), and almost no AVOR was observed in any goldfishtested under these rearing conditions. Among these species,adult AVOR performance has only been measured fully ingoldfish, which, unlike larval animals, exhibit a robust eyevelocity in response to angular vestibular stimulation even atlow accelerations (Pastor et al. 1992; Schairer and Bennett1986a).

Based on existing anatomical and physiological data of innerear development in zebrafish (Haddon and Lewis 1996; Whit-field et al. 2002) as well as the nearly ubiquitous presence of anAVOR in the juveniles of other vertebrates, e.g., human (Fin-occhio et al. 1991), kitten (Flandrin et al. 1979), and chick(Wallman et al. 1982), the total lack of an AVOR in earlylarval fish was unexpected. The semicircular canals are formed,replete with hair cells in the cristae, by 3 dpf (Haddon andLewis 1996; Waterman and Bell 1984). By 5 dpf, the canalshave attained their basic configuration (Bever and Fekete2002), and afferents fibers from the cristae have entered thelarval brain stem (Haddon and Lewis 1996; Raible and Kruse2000). Likewise, vestibulo-oculomotor projections also appearat these early stages (Suwa et al. 1996). Whether the hair cellsfrom the canal cristae are functional or not is unknown;nevertheless, hair cells from zebrafish lateral line neuromastsare operational by 5 dpf (Nicolson et al. 1998). Otolith haircells and afferents must be active if static VOR behavior, asmeasured in the light, in 5 dpf zebrafish (Moorman et al. 1999)

can also be shown in the dark; in addition, proper otolithactivity is important both for survival (Riley and Moorman2000) and posture (Nicolson et al. 1998; Riley and Moorman2000) of young larvae. Despite the presence of all the consti-tutive parts, the absence of behavioral manifestations of canalactivity may be due to an inability of inertial forces to driveendolymph flow within the canals of larval animals.

This hypothesis is consistent with theoretical studies ofvertebrate semicircular canal morphology (Muller 1994,1999; Rabbitt 1999; Rabbitt et al. 2003). Endolymph move-ment within the canals, and hence acceleration sensitivity, issignificantly constrained by the canal arc radius as well asfrictional forces induced by the canal lumen diameter.Indeed, acceleration sensitivity drops as the square root oflumen diameter; thus, for example, the canal sensitivity oflarval carp has been calculated to be 160 times less than thatof an adult guppy, Lebistes (Muller 1999). If canal lumendiameter is the correct explanation for the low AVOR gains,it appears that, even in the largest cyprinid larvae studied,endolymph flow continues to be significantly less than thatof the adult animals. Nevertheless, when the canals dobecome sensitive to acceleration stimuli as in large medaka(Fig. 11, C and D), the vestibular time constant was foundto be short (�1s; Fig. 12D). In contrast, the vestibular timeconstant of adult animals is several seconds in length: 5 s intoadfish (Boyle and Highstein 1990), 3 s in goldfish (Hart-mann and Klinke 1980), and 6 s in primates (Fernandez andGoldberg 1971). In addition to vestibular sensitivity, mod-eling studies have suggested that size also affects the lengthof the vestibular time constant, with � proportional to thearea of the cupula (Rabbitt et al. 2003).

Thus it seems possible that the lack of an AVOR in larvalfish has less to do with genetic expression of the necessarycentral neurons and more to do with simply the very small sizeof newly hatched larvae. This hypothesis is testable as activa-tion of the angular vestibular system, either through mechan-ical or chemical stimulation, and the measured behavioralresponse could provide evidence that the central connectivity ispresent and operational despite the apparent lack of endolymphflow. Alternatively, the big ears zebrafish mutant identified inthe Tubingen screen with large semicircular canals (Whitfieldet al. 1996) or another teleost species, such as rainbow trout(Oncorhynchus mykiss), that hatches at a larger size mightpresent unique opportunities to establish the correlation be-tween canal size and acceleration sensitivity.

In summary, the precocious optokinetic responses and neartotal lack of angular vestibular activity in larval fish suggeststhat evolutionary pressures have selected for a highly func-tional visual system over the requirement to sensitively detectangular acceleration. Because the animals tested exhibit differ-ing lifestyles from highly kinetic zebrafish to slowly swimminggoldfish with medaka intermediate in behavior, it is possiblethe small variations in the behaviors observed could be due toslight differences in their ontogenetic timing. Indeed, onemight hypothesize that any small, free swimming larvae, nomatter how primitive or derived, would follow a similar patternof oculomotor development. Therefore zebrafish and medakaoffer excellent models for genetic dissection of the neuronalcomponents underlying oculomotor behavior.



A C K N O W L E D G M E N T S

The authors thank G. Gamkrelidze and I. Netravali for participation duringpreliminary experiments and D. Chu for support with fish care and mainte-nance.

G R A N T S

This work was supported by grants from the National Institutes of Health toD. W. Tank and R. Baker and a National Research Service Award from theNational Eye Institute to J. C. Beck.

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