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Thomas R. Consi*, M. Beatrice Passani and Eduardo R. Macagno **. Department .... inside surface of the cone (180 ) was painted matte silver; the stimulus was ...
J Comp Phyisol A (1990) 166:411-420

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Eye movements in Daphnia magna Regions of the eye are specialized for different behaviors T h o m a s R. Consi*, M. Beatrice Passani and E d u a r d o R. M a c a g n o ** Department of Biological Sciences, Columbia University, New York, NY 10027, USA Accepted September 3, 1989

Summary. Three types of behavior of the compound eye o f Daphnia magna are characterized: 'flick', a transient rotation elicited by a brief flash of light; 'fixation', a maintained eye orientation in response to a stationary light stimulus of long-duration; 'tracking', the smooth pursuit of a moving stimulus. The magnitudes of the flick and fixation responses vary with stimulus position and are generally proportional to stimulus intensity, although at high intensities there is an attenuation of both behaviors. When the stimulus is placed at a position ,-, 80 ~ dorsal to the eye axis, there is no response; this area is called the null region. For stationary stimuli in other positions, the direction of the response is such as to bring the stimulus closer to the null region. During tracking, the relative positions o f the eye and stimulus change; the eye velocity is approximately half that of the moving stimulus. The regions o f the eye in which these behaviors may be induced are different, being largest for flick and smallest for tracking. It is proposed that flick and fixation responses are a means for rotating the eye so that the stimulus is within the area surrounding the null region which is used for tracking. Key word: Visual system - C o m p o u n d eye - Oculomotor responses - Arthropod

Introduction The single, medially located compound eye of the small crustacean Daphnia magna is highly mobile and displays a variety o f movements in response to visual stimuli (Frost 1975). The underlying neuronal circuitry responsible for the transformation of visual input to eye m o t o r commands is relatively simple and amenable to detailed cellular and ultrastructural study (Macagno et al. 1973; * Present address: Sea Grant Program, Massachusetts Institute of Technology, Cambridge, MA 02139, USA ** To whom correspondence should be addressed

Sims and Macagno 1985; Consi et al. 1987). It is thus of great interest to understand the relationship between the characteristics of the stimulus and the corresponding behavioral response in order to comprehend the functional capabilities o f this circuitry. Eye movements in Daphnia have been the subject of several previous studies (reviewed by Fraenkel and Gunn 1961; Wehner 1981). In most of these studies the spatial relationship between the stimulus and the array of photoreceptors in the eye were either not known or only roughly determined (e.g., dorsal quadrant of the eye). In the work reported here, we recorded simultaneously the positions o f the eye and the stimulus for both static and moving stimuli. We studied 3 classes of responses: flick, a quick transient rotation in response to a flash o f light (Consi and Macagno 1985); fixation, a maintained positional change in response to a stimulus of long duration; tracking, the pursuit of a moving stimulus. The terms fixation and tracking are used here to denote specific responses o f the Daphnia compound eye, analogous to but somewhat different from the mammalian eye responses to which these terms are commonly applied. The stimulus used in these experiments was a narrow stripe of white light oriented and positioned so as to stimulate only the 4 pairs of medially located ommatidia. This stimulus elicited rotations predominantly in the dorsoventral direction, thus simplifying the analysis o f the motion of the eye. Which of the 4 pairs o f ommatidia were stimulated, and thus the inputs to the oculomotor system, were determined for each of the behaviors studied.

Materials and methods Animals. Only large (3-4 mm long) adult females were used in these experiments. The animals were from a clonal population maintained continuously in our laboratory for several years (see Consi and Macagno 1985, for maintenance protocols). Behavioral apparatus. The apparatus used for the generation and observation of eye movements was a modified version of the appa-

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Fig. 1. Simplified diagram of the apparatus used to observe Daphnia eye movements. ! 1000 W xenon arc lamp; 2 condenser lens; 3 filter holder; 4 focusing lens; 5 electronic shutter; 6 recollimating lens; 7 paper mask used to form wedge-shaped stimulus; 8 ground glass screen; 9 optical image rotator; 10 projection lens; 11 first surface mirror; 12 plastic cone, inside painted silver (180 ~) and fiat black (180~ 13 water-filled tube with daphnid inside glued to a pin and held in place with a micromanipulator; 14 compound microscope; 15 infrared sensitive video camera. Inset: Daphnia eye (viewed from the right) and screen coordinate systems. A - K lenses overlying the 11 ommatidia on the right side of the eye (the corresponding ones on the left side not shown); PA photoreceptor axons. In the diagram the eye and conical screen coordinates are aligned, and the degree markings apply to both cordinate systems. Eye axis bisects the eye at 0 ~ ratus used in earlier experiments on Daphnia eye flick (Consi and Macagno 1985). A simplified schema of the apparatus is presented in Fig. 1. A daphnid was fixed with cyanoacrylate glue to a pin attached to a micromanipulator. It was then positioned, on its side, in the center of a cylindrical, water-filled glass chamber coaxial with and centered within a truncated plastic cone. Half of the inside surface of the cone (180 ~) was painted matte silver; the stimulus was projected onto this area (the conical screen). The other half of the cone was painted fiat black. The visual stimulus was a vertical stripe of white light 10~ wide and 60 ~ long generated as follows. Broad-band (white) light from a 1000 W xenon arc lamp (Photochemical Research Associates, London, Ontario, Canada) was collimated onto a ground glass screen through a black paper mask with a 10~ wide pie-wedge cut out of it. After passing through an optical image rotator (C & H Sales Co., Pasadena, Calif., USA, stock no. OL8201), the light wedge was projected onto the conical screen, where it formed the vertical stripe. The stimulus seen by the Daphnia was a light stripe extending 10~ in the dorsoventral direction and 30 ~ to each side of the medial plane of its compound eye (ignoring refraction by the water and the glass of the chamber). These parameters of the stimulus were chosen so as to stimulate only the pairs of ommatidia which straddle the median plane (ommatidia A, B, C, and D; see Fig. 1, inset). Movement of the stripe on the silvered portion of the conical screen was accomplished with the image rotator. The stimulus was gated with an electronic shutter (Vincent Associates, Rochester, NY, USA, Model 26), and the light intensity was modulated with glass neutral density filters. Light intensity (in lux) was measured with a photodiode (EG & G, Salem, Mass., USA, silicon photodiode, HUV-2000B) calibrated with a photome-

ter (EG & G, model 550). The measurements were made at the position of the tethered animal in the glass chamber, but without water. Eye movements were observed using a video camera (Dage MTI, Michigan City, Ind., USA, model 65 with a Nuvicon tube) coupled to a compound microscope and recorded on magnetic tape with a video cassette recorder (Panasonic 8950). The total magnification of the system from specimen to image on the video monitor was about 300 x . For observation, the animal was transilluminated with infrared light generated by a filtered strobe lamp (General Radio Co., Concord, Mass., USA, 1539-A Stroboslave, with a Kodak Wratten 87C filter). At 780 nm, the absorbance of this filter is 3 log units; extrapolated to 700 nm, the absorbance is approximately 13 log units. At this wavelength the sensitivity of the Daphnia eye is about 2 log units below its maximum at ~530 nm (Consi and Macagno 1985). As expected, we saw no response whatsoever by the animal to this illumination. The strobe lamp was synchronized with the vertical sync pulse of the video camera; this technique yielded the sharpest video image. A microcomputer (Commodore 64) served as the central coordinator of the apparatus, controlling the electronic shutter, the infrared illuminator, and the video cassette recorder. The stimulus position was monitored with a potentiometer geared to the image rotator and read by the microcomputer via an 8 bit A/D converter. A video title generator (FOR-A, Co., Los Angeles, CA, USA, TG-160), also controlled by the microcomputer, added the video frame number, stimulus position, and shutter status (on/off) to each video frame.

Experimental protocols. Before beginning an experiment, the animal was dark-adapted in the apparatus for at least 30 min. The results presented here were obtained from a total of 23 animals; each of the phenomena described was studied in several animals. The Daphnia was always mounted with its right side up, facing the observer. To control for any left-right asymmetry of the stimulus, a fixation experiment was performed in which the animal was mounted with its left side facing the observer. The results from this experiment were identical to the results from experiments with the animals mounted with their right sides facing the observer. For the eye flick experiments, the duration of the stimulus was 67 ms or less, which is less than the estimated latency of the response ( > 100 ms). Intensity/response relationships were obtained by presenting stimuli of increasing intensities at a constant position. At each intensity, a series of 3 or 5 pulses was given with an interpulse interval of 5 or 10 s; longer intervals between series of pulses (15 s to 5 min) were used in order to minimize possible contributions of light adaptation. Intensity/position relationships were determined by repeating the experiments at different stimulus positions. Alternatively, a fixed intensity was selected, and the stimulus was presented at various positions around the eye. This procedure was then repeated for other intensities. Essentially the same protocol was used to examine fixation but with stimulus durations of 0.5, 1, 4, and 5 s. The tracking response was obtained by moving the 10~ wide wedge of light around the tethered animal. The stimulus was either continuously on while it was moved or it was pulsed at a frequency of 5 Hz (100 ms on, 100 ms off). In some experiments the stimulus was moved at a fairly constant velocity over a range of +65 ~ from the center of the conical screen. In others, the stimulus was oscillated approximately +14 ~ about a specific position. The stimulus velocities used ranged from 10~ to 25~ Light intensities used in the tracking experiments were those which elicited large eye flick responses. Data analysis. To measure eye position, we defined two polar coordinate systems: eye-centered coordinates and screen-centered coordinates (Fig. 1, inset). The origin of the eye-centered coordinate system was approximately at the center of rotation of the eye. A line bisecting the eye and perpendicular to the flat side where the photoreceptor axons exit was defined as the eye axis (0~ Posi-

Th. R. Consi et al. : Eye movements in Daphnia magna tions dorsal to the eye axis were positive, ventral were negative. The screen-centered coordinate system was concentric with the eyecentered coordinates. In all experiments the animal was positioned with its eye axis pointing in one of five directions with respect to the screen prior to stimulation: 0~ + 45 ~ or ___90~ For the flick and fixation experiments the stimulus was always placed within ___23~ of the center of the screen. Such a procedure was necessary to minimize intensity variation with stimulus position due to small optical inhomogeneities in the system. Video tapes of the eye movements were analyzed frame by frame to obtain eye angle vs. time data. A cross pattern was mounted in the apparatus at the animal's position and aligned with the screen coordinate system. The pattern was displayed on the video monitor, and the coordinates were drawn on the monitor screen. Next, a tracing of the eye was made from a freeze-frame image onto a sheet of clear acetate which was mounted onto the protractor end of a drafting arm. The arm was arranged so that it moved in a plane parallel to the monitor screen. The tracing was superimposed on the first frame, and the angle between the eye axis and the 0 ~ coordinate on the conical screen was measured. The video tape was advanced to the next frame and the process repeated. The time resolution of the eye movement data was the video frame duration of 33 ms. The protractor end of the drafting arm was coupled to a potentiometer which was read by the microcomputer via the analog to digital converter. The computer converted the potentiometer voltage readings into eye angles. Repeated tracings of the same eye movement yielded sets of values which were repeatable to within ___3~ Frame number and stimulus position were read from the video image and stored with the corresponding eye angle. In each file of eye movement data, the average position of the eye axis (in screen coordinates) in the dark, prior to stimulation, was determined by inspection. This angle was subtracted from all eye angles and corresponding stimulus positions so that a graph of eye and stimulus position vs. time shows the eye at an average prestimulus position of 0~ (see Fig. 1, inset). In such graphs, therefore, the ordinate was labeled 'position in adjusted screen coordinates' (position on the conical screen minus prestimulus position).

Results Eye flick In the dark, the compound eye displayed an irregular j i t t e r o r t r e m o r o f s m a l l a m p l i t u d e (see e y e m o v e m e n t w a v e f o r m s p r e c e d i n g e v o k e d r e s p o n s e s , F i g . 2). I n res p o n s e t o b r i e f (67 m s ) p r e s e n t a t i o n s o f t h e l i g h t s t r i p e (see M a t e r i a l s a n d m e t h o d s ) , t h e d a r k - a d a p t e d e y e p e r f o r m e d t r a n s i e n t r o t a t i o n s o r flicks ( F i g . 2). Both the direction and the amplitude of the eye flick depended upon the position of the stimulus on t h e e y e ( F i g . 3). S t i m u l i p l a c e d in a n a r e a 7 0 ~ ~ d o r s a l t o t h e e y e a x i s g e n e r a l l y f a i l e d t o elicit a s i g n i f i c a n t r e s p o n s e a t m o s t i n t e n s i t i e s in a n y o f t h e 7 a n i m a l s f o r w h i c h w a v e f o r m s w e r e o b t a i n e d o r in s e v e r a l others for which responses were visually analyzed. H e n c e , w e t e r m e d t h i s a r e a o f t h e e y e t h e null r e g i o n . S t i m u l i p o s i t i o n e d d o r s a l t o it p r o d u c e d e y e r o t a t i o n s in a d o r s a l d i r e c t i o n , w h i l e s t i m u l i p o s i t i o n e d v e n t r a l ly p r o d u c e d v e n t r a l r o t a t i o n s . T h e a m p l i t u d e o f v e n t r a l l y d i r e c t e d flicks i n c r e a s e d a s t h e s t i m u l u s w a s m o v e d f r o m 70 ~ t o 0 ~ w h e r e a m a x i m u m r o t a t i o n occurred, and then decreased as the stimulus was

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Fig. 2A, B. Representative eye flick waveforms. A Ventrally directed eye rotations in response to 67-ms light pulses of increasing intensity placed approximately 45 ~ dorsal to the eye axis. i log intensity in lux (log I) = -2.35 ; ii log I = - 1.31 ; iii log I = - 0.0531. B Dorsally directed eye rotations in response to 67-ms stimuli placed approximately 115~ dorsal to the eye axis. i log I = - 1.68; ii log I = - 0 . 5 0 9 ; iii log I=0.330. Ordinate, position in adjusted screen coordinates (see Materials and methods). Dotted line represents stimulus position; up arrow, stimulus on; down arrow, stimulus off. Insets: relative position of the stimulus (darkened arc) on the eye at the onset of stimulation

m o v e d o n . T h e o v e r a l l s h a p e s o f t h e a m p l i t u d e vs. stimulus position curves were the same at the intensities t e s t e d . A comparison of the responses at different intensities ( F i g . 3 A ) i n d i c a t e s t h a t t h e v e n t r a l flicks inc r e a s e d in size w i t h i n t e n s i t y u p t o a m a x i m u m a n d t h e n d e c r e a s e d a t h i g h e r i n t e n s i t i e s ( F i g . 3 B). T h e response amplitude generally increased with intensity over a range of about 3 log units, flattened out, and then decreased at higher intensities.

Fixation Fixation occurred when the eye was presented with a long duration (>200 ms) stimulus positioned on c e r t a i n r e g i o n s o f t h e eye ( F i g . 4). I n r e s p o n s e t o a ventral stimulus, the eye rotated ventrally, maintained

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Fig. 3A, B. Dependence of the eye flick upon stimulus position and intensity. A Maximum eye rotation vs. stimulus position during flicks elicited by 67-ms light pulses at increasing light intensities: (..) log I = - 2 . 2 5 ; ( . . . . . ) l o g i = - 1 . 2 3 ; (--) logI=0.175; ( - - ) log I = 0.837. On the abscissa, the position of the stimulus on the eye prior to stimulation (in eye coordinates) was calculated by averaging the eye positions in 10 video frames (333 ms) prior to stimulation. B Maximum eye rotation vs. stimulus intensity during eye flick. Stimuli were placed at either 0~ ~ (solid line) or 100~ ~ (dashed line) dorsal to the eye axis. In both A and B data from one animal, each point representing average of responses to 5 consecutive light pulses (in A standard deviations omitted for clarity); positive and negative values on the ordinate represent, respectively, dorsal and ventral rotations

a nearly constant position and, once the stimulus was turned off, rotated back, generally overshooting its original position before returning to it (Fig. 4A). Dorsally directed fixations (Fig. 4B) showed similar features but were characterized by slower rotations to and from the fixation position as well as the lack of an overshoot following the offset of the stimulus. During fixation in either direction, the eye continued to show the small-amplitude oscillations which were present before stimulation. Both the magnitude and direction of fixation varied with the position of the stimulus, as illustrated in Fig. 5 A. Examination of this graph reveals some close similarities between fixation and flick. For both behaviors, little or no response occurred when the stimulus was placed in the null region. Stimuli placed dorsal to this region elicited dorsally directed fixations, while stimuli placed ventral to it caused re-

Fig. 4. A Ventrally directed response to a 4-s stimulus located at 0 ~ on the eye (horizontal dashed line); log I = - 1.22. B Dorsally directed fixation response to a 4-s stimulus initially at about 125 ~ (horizontal dashed line); log I = - 1 . 2 2 . Eye position is in adjusted screen coordinates. Up arrow, stimulus on; down arrow, stimulus off. Insets: relative position of the eye and the stimulus (darkened arc) at onset of stimulation

sponses in the ventral direction. Stimuli placed near the eye axis caused maximum responses. Ventral fixations, however, did not occur over the same range of stimulus positions as flicks. No stable fixations occurred when stimuli were placed more ventral than approximately - 3 0 ~ (cf. Fig. 3 A with Fig. 5A). The eye failed to maintain a constant position after an initial transient, oscillating irregularly or holding unpredictable positions for short times. Another way of displaying the fixation data is to plot the initial stimulus position vs. the stimulus position during fixation (final position) in eye-centered coordinates (Fig. 5 B). This curve shows a nearly linear relationship between initial and final positions in the 110 ~ to 0 ~ region, with a slope of about 0.6. A slope of 1.0 would have indicated no fixation, while a slope of zero would have indicated fixation of the stimulus on a specific region of the eye. It is also evident from an examination of this graph that the result of either dorsal or ventral fixation was a displacement of the stimulus toward the null region of the eye. The relationship between stimulus intensity and response amplitude for ventrally directed fixations was found to be similar to that for eye flicks (Fig. 6). The amplitude of rotation first increased with stimulus intensity up to a plateau and then decreased at

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Fig. 6. Fixation rotation plotted as a function of stimulus intensity for two different animals. Stimulus duration, 500 ms. The fixation rotation is expressed as eye position during fixation minus prestimulus eye position, where prestimulus eye position is the average of eye positions during 15 prestimulus video frames (500 ms). Eye position during fixation is the average in the 5 video frames (167 ms) following the first l0 video frames (333 ms) after the onset of stimulus, so chosen to avoid initial transients. Each point is the average of values from 3 responses; vertical bars, standard deviations. Solid line, animal A, initial stimulus position approx. 13~ dashed line, animal B, initial stimulus position approx. 11~

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traced every 5 video frames (167 ms) starting 1 s prior to the stimulus. Ordinate, magnitude of eye rotation, calculated as eye position during fixation minus initial eye position. Eye position during fixation is the average eye position in 12 traced frames (2 s) during stimulation, 1 s after the onset of the stimulus (to eliminate initial transient motion); the initial eye position is the average eye position of 6 traced video frames (a time period of 1 s, every 5th frame traced) prior to stimulus. Abscissa, initial stimulus position on the eye. Data from 1 animal; each point is the average of values from 4-11 (mostly 5) stimulus pulses given as trains. B Plot of initial vs. final stimulus position on the eye (solid line). The data used are those presented in A. Dashed line, plot for no fixation behavior; the position of the stimulus on the eye is the same before and during fixation. Shaded areas indicate the null region of the eye. Note that the data curve crosses the dashed diagonal at approximately 86~ on the eye, in the null region where no response normally occurs. Ordinate, final stimulus position on eye, calculated as stimulus position minus eye position during fixation; abscissa, same as in A

h i g h e r intensities. W h i l e significant a n i m a l to a n i m a l differences in r e s p o n s e a m p l i t u d e w e r e seen, especially at the intensities w h i c h elicited m a x i m u m responses, the overall s h a p e s o f the r e s p o n s e c u r v e s were similar f o r all the a n i m a l s t h a t w e r e tested.

A possible e x p l a n a t i o n f o r the u n e x p e c t e d a t t e n u a t i o n o f f i x a t i o n a n d flick at h i g h intensities c o u l d c o n c e i v a b l y h a v e b e e n light s c a t t e r i n g in the a p p a r a tus. F o r e x a m p l e , while the stimuli w e u s e d in these e x p e r i m e n t s were d e s i g n e d t o stimulate o n l y single bilateral pairs o f m e d i a l o m m a t i d i a , at h i g h intensities several o m m a t i d i a m i g h t h a v e b e e n s t i m u l a t e d simult a n e o u s l y b y scattered light, l e a d i n g t o a n t a g o n i s t i c i n t e r a c t i o n s t h a t r e d u c e d the r e s u l t a n t flick o r fixation. To test this possibility, c o n t r o l f i x a t i o n experim e n t s were p e r f o r m e d w i t h a p a p e r m a s k a r o u n d the s p e c i m e n c h a m b e r w h i c h served t w o f u n c t i o n s : to collimate the s t i m u l u s a n d p r e v e n t o b l i q u e light r a y s f r o m s t i m u l a t i n g n e a r b y o m m a t i d i a , a n d to eliminate a n y possible s e c o n d a r y reflections o f the s t i m u l u s o n the screen f r o m r e a c h i n g the eye. T h e p r e s e n c e o f the m a s k , h o w e v e r , m a d e n o o b s e r v a b l e difference in the results, a n d a t t e n u a t i o n o f the f i x a t i o n res p o n s e s w a s still o b s e r v e d at high light intensities.

Tracking T h e eye f o l l o w e d the s t i m u l u s in p h a s e w h e n the stimulus w a s d i s p l a c e d slowly b a c k a n d f o r t h o v e r the d o r s a l h a l f o f the eye (Fig. 7). To e x p l o r e the spatial b o u n d a r i e s o f this t r a c k i n g response, we m o v e d the s t i m u l u s o v e r large angles (Fig. 8). T h e eye w a s able to t r a c k o n l y if the s t i m u l u s w a s in a r e g i o n o f the eye f r o m a p p r o x i m a t e l y 120 ~ to 20 ~. In the case

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shown in Fig. 8A, the eye made a dorsally directed rotation (moving the null region toward the stimulus) when the stimulus reached about 153 ~ on the eye. This rotation moved the stimulus to ,-,110 ~ on the eye, at which point the eye began to track the ventrally moving stimulus (Fig. 8 A, left arrowhead). When the direction of the stimulus was reversed at ,-~60 ~ on the eye, the eye also reversed direction and tracked the stimulus in the dorsal direction until the stimulus has slipped to ,~ 120 ~ on the eye (Fig. 8A, right arrowhead). The eye then lost the stimulus and rotated back to its dark-adapted position. In Fig. 8 B, the eye tracked the stimulus until it moved beyond ,-~ 19 ~ (left arrowhead), at which time the eye rotated to and remained in its dark-adapted position. After the stimulus reversed direction and moved past ,-, 18 ~ the eye began tracking again (Fig. 8 B, right arrowhead). Figure 8C shows that when a stimulus was moved in the ventral half of the eye, the eye displayed large oscillations and failed to track. To determine whether or not the spatial relationship between the eye and the stimulus was different for tracking in the dorsal and ventral directions, the data from Fig. 8A were replotted as a graph of stimulus position on the eye vs. stimulus position on the conical screen (Fig. 9A). While the stimulus was in the tracking zone (dashed lines), the eye and the stimulus had the same angular relationship. When a flashing stimulus (5 Hz; 100 ms on, 100 ms off) was moved across the dorsal area of the eye, the eye followed the stimulus with a 5 Hz flick of small amplitude superimposed on the tracking response (data not shown). We did not determine in these experiments the maximum or minimum stimulus velocities that the Daphnia eye can track. While the eye was able to follow the stimulus readily at the velocities we used in these experiments, it

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Fig. 7. Representative waveform o f tracking eye movement. Stimulus was centered at about 40 ~ in the dorsal region of the eye and moved by hand in the dorsoventral direction with a maximum excursion of about ___14 ~ The maximum excursion of the eye was about _+8.5 ~ e, eye trace; s, stimulus trace

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Fig. 8 A - C . Tracking waveforms. Positions of the moving stimulus on the eye are illustrated in the insets. A Stimulus was moved in the dorsal half o f the eye; the eye picked up and followed the stimulus at the left arrowhead and lost the stimulus at the right arrowhead. A least squares linear regression was performed on the sections of eye and stimulus traces bounded by the vertical lines in order to obtain the eye and stimulus speeds (slopes of the regression lines). B Stimulus was moved dorsoventrally across the eye axis. The tracking movement was lost when the stimulus swept across and past 40 ~ in the ventral region (eye movement trace delimited by arrowheads). C Eye responses to a stimulus moving in the ventral half of the eye. No tracking behavior is apparent in this tracing; in fact, the eye is initially in a ventral fixation position which is soon lost (trace within the arrowheads). Stimulus intensity, log I ~ - 0 . 2 1 ; data from one animal, e, eye trace; s stimulus trace

did so at a slower angular velocity. In Fig. 8A, in the region of the curve between the vertical lines, the average speed of the stimulus was 14.9~ while the eye followed at an average speed of 7.3~ The eye was generally found to move 2-3 times slower than the stimulus for stimulus speeds of 10~ to 25~ The small-amplitude irregular oscillations characteristic of the eye in the dark continued during tracking.

Discussion

Specialization of eye regions for different behavioral responses The areas of the medial region of the eye over which a small light stimulus can elicit the 3 responses overlap one another but have somewhat different extents.

Th. R. Consi et al. : Eye movements in Daphnia magna

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Fig. 10. Behavioral regions on the Daphnia compound eye. Each arc represents the region of the eye in which a stimulus can elicit the indicated response. Null region is shaded (70~176 Eye flick and fixation stimuli dorsal to the null region induced dorsal (counterclockwise in this diagram) eye rotations, and stimuli ventral to the null region induced ventral (clockwise) eye rotations. Dots indicate that the dorsal boundaries of the regions are undetermined. The eye-centered coordinate system is shown surrounding the eye; anterior is at 0~ dorsal is at 90~ Drawing of the eye made from a computer reconstruction (see text); A-K, lenses of the eye; PA, photoreceptor axons; flick, eye flick; fix, fixation; track, tracking

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Fig. 9A, B. Stimulus position on the conical screen vs. stimulus position on the eye during tracking, flick, and fixation responses. A Curve of the tracking response. Note the linear relationship

between the stimulus on the screen and its corresponding position on the eye while the stimulus was in the tracking zone (region between the outer dashed horizontal lines), v, ventral tracking; d dorsal tracking. B Curves for eye flick (-.-) and fixation (-El-) responses. Y-coordinate of each point on the eye flick curve denotes the position the stimulus would have been on the eye (had it been on) when the eye was at its maximum rotation during the response. Y-coordinate of each point on the fixation curve indicates the stimulus position on the eye during fixation. Unconnected dots are the points from the tracking curve in A. Data in tracking curve from the experiment illustrated in Fig. 8 ; eye flick curve calculated from Fig. 3A (log I = -0.175); fixation curve data from experiment illustrated in Fig. 5. Ordinate, stimulus position on eye in eye coordinates; abscissa, stimulus position on the conical screen in adjusted screen coordinates; region between the two outer dashed horizontal lines is the tracking zone; area between the two inner dashed lines is the null region As determined f r o m position/response measurements, a stimulus can induce a flick if it is placed a n y w h e r e f r o m 120 ~ to - 9 0 ~ on the eye a n d fixation when located anywhere f r o m 120 ~ to - 3 0 ~ o n the eye, with the exception o f the null region, where neither o f these responses occurs. The d o r s a l - m o s t b o u n d a r i e s o f the eye flick a n d fixation areas have n o t been accurately

determined, a n d they m a y well extend further t h a n 120 ~ (some o f o u r tracking data suggest this). Tracking occurs in response to a stimulus when it is m o v i n g in the region f r o m 120 ~ to 20 ~ o n the eye. These relationships are d i a g r a m m e d in Fig. 10. The use o f a small stimulus in these experiments permits us to define the position o f the stimulus o n the eye fairly accurately. Two additional pieces o f inf o r m a t i o n allow us to interpret this position in terms o f the o m m a t i d i a which are principally stimulated. One involves the size and shape characteristics o f the receptive fields o f each o m m a t i d i u m ( Y o u n g and D o w n i n g 1976; Y o u n g 1981). Calculations by these researchers f r o m optical m e a s u r e m e n t s o f individual lenses yielded receptive fields a b o u t 40 ~ in diameter with central zones o f 100% sensitivity a b o u t 10 ~ in diameter and steep sides. Y o u n g a n d D o w n i n g also c o n c l u d e d that the receptive fields o f neighboring o m matidia overlap only slightly, and that there should be only small or no blind gaps between them. Given these estimates and the size o f the stimulus we used, we can conclude that, m o s t o f the time, single bilateral pairs o f medial o m m a t i d i a were stimulated in o u r experiments. The second i m p o r t a n t fact is the relative placement o f the o m m a t i d i a in the eye. This we have obtained f r o m a 3-dimensional c o m p u t e r reconstruction o f a fixed adult Daphnia eye m a d e f r o m serial sections (Schehr 1984). The d r a w i n g o f the eye in

418

Fig. 10, showing the location of the lenses, each corresponding to an ommatidium, was derived from a side view of the computer reconstruction. The medial ommatidia are those labeled D, A, B, and C, from dorsal to ventral. In light of these considerations and our determination of the areas of the eye involved in the generation of the movements studied, we can make behavioral assignments to the medial ommatidia. Eye flick can be elicited by a flash of light that individually stimulates any of the ommatidia D, A, B, or C. Stable fixation occurs when the stimulus is located within the receptive fields of ommatidia D, A, or B. The ventral half of the receptive field of ommatidium B and the dorsal half of the receptive field of C lie in the area in which partial fixation along with large rotations and oscillations have been observed during stimuli of long duration. The null region coincides with the dorsal half of the receptive field of ommatidium D. The tracking zone includes the receptive fields of ommatidia D and A. All 3 behaviors, in addition, can be elicited by stimuli dorsal to the receptive field of ommatidium D. The constant state of oscillation (tremor) exhibited by the eye (Frost 1975) could serve to extend the receptive field of ommatidium D somewhat further dorsally and so provide the input to the oculomotor system for more dorsally placed stimuli. Alternatively, the receptive field of ommatidium G may extend medially enough to provide the input signal. It should be noted that this assignment of behavioral roles to specific ommatidia is only approximate, since the exact orientations of the ommatidial axes in the eye have not been determined in a live preparation, and the computer reconstruction of a fixed and sectioned eye is necessarily somewhat distorted by the shrinkage entailed in the histological procedures. Behaviorally specialized eye regions are well documented in the compound eyes of arthropods (e.g., Collett and Land 1975, flies; Nalbach and Nalbach 1987, crabs). Typically, these specialized regions correspond to anatomically distinct areas of the eye in which the ommatidia are optimally constructed for the particular behavior. For example, the foveal region of the male fly contains a flattened array of ommatidia with small interommatidial angles which give this area high spatial acuity (Collett and Land 1975). This situation holds even for the related cladoceran Polyphemus pediculus which has several anatomically distinct regions in its single compound eye (Nilsson and Odselius 1983; Odselius and Nilsson 1983). The centrally placed acute zone of this eye may be involved in small object tracking (Young and Taylor 1988; Young 1988). The eye ofDaphnia magna is unusual in that it is composed of a fairly uniform array of ommatidia (Flaster et al. 1982), and the behaviorally distinct regions are functionally defined but not anatomically apparent.

Th. R. Consi et al. : Eye m o v e m e n t s in Daphnia magna

Some characteristics of the flick and fixation do not reflect known properties of the Daphnia photoreceptors Daphnia photoreceptors, like those of other invertebrates, show a sigmoid dependence of the peak depolarization with stimulus intensity (Schehr 1984). The intensity range from threshold to saturation is 3-4 log units and similar to that for the eye movements discussed here (Figs. 3B, 6). At higher intensities, however, eye movements do no reflect the response characteristics of the photoreceptors. For brief light flashes the peak of the photoreceptor potential saturates and remains fairly constant at high intensities. The eye flick shows a graded decrement as the light intensity is increased beyond a 'peak response' value. Clearly, if the Daphnia nervous system is reducing the size of the output to the eye muscles, then the behavioral response decrement must be occurring at subsaturation intensities in order for the Daphnia nervous system to ' k n o w ' the light intensity. For stimuli of long duration, the magnitude of the receptor potential following the initial transient decreases as the photoreceptors adapt (Schehr 1984). This adaptation may be a cause of the reduced fixation responses seen at high intensities. Unfortunately, the behavioral and physiological experiments discussed here are not directly comparable because different stimuli and experimental protocols were used. To determine the exact relationship between the photoreceptor response characteristics and the eye movements, such a set of coordinated experiments is needed. Another explanation for the reduction in eye excursion seen at high intensity might be scattered light entering additional ommatidia and producing an antagonistic response. Such effects were observed when two stimuli which induce opposite eye movements were presented simultaneously (Consi et al. 1987). Control experiments eliminated the possibility that light scattering in the apparatus itself was the source of the high intensity eye behavior (see Results). Light scattering between rhabdoms after entering the eye is probably not significant; Daphnia photoreceptors are filled with screening pigment granules which surround the rhabdomeres and do not migrate in response to changes in illumination (R6hlich and T6r6 1965) as they do in other species (e.g., Shaw and Stowe 1982). We are left with the possibility that light scattering occurs within the head but external to the eye (e.g., on the carapace or the gut), which cannot be completely eliminated by the experiments reported here.

Eye movements and Daphnia behavior A daphnid will orient in an illuminated column of water with its dorsal side facing the direction of the incoming light. This behavior is known as the dorsal

419

Th. R. Consi et al. : Eye movements in Daphnia magna

light reaction, and it has been identified and studied in both Daphnia and a variety of other arthropods (for reviews, see Fraenkel and Gunn 1961 and Wehner 1981). Harris and colleagues (Harris and Wolfe 1955; Harris and Mason 1956) found that the compound eye was necessary for proper orientation during the day and for the daytime component of the vertical migration pattern. The most prominent underwater visual feature is Snell's window, the circle of light entering the water from the sky caused by the critical angle of light refraction at the air-water interface (Lythgoe 1979). The edge of Snell's window forms a constant and prominent contrast boundary to which an underwater animal can orient. Ringelberg and colleagues (Ringelberg 1964; Ringelberg et al. 1975) have studied the orientation of Daphniato static angular light distributions which mimic the effect of Snell's window. When presented with such a stimulus the daphnid made an eye rotation which was followed by a body movement, and the animal assumed a stationary position. In this oriented position the dorsal region of the compound eye was pointed at one of the contrast boundaries. In addition, the area of the eye which became fixated at the contrast boundary was a dorsal region which was roughly the same as the tracking zone which we have identified. The oculomotor system of Daphnia appears to be part of a body orientation control system: the movable eye locates a contrast, and the body rotates to bring the eye and body into some preferred position. This idea has been proposed by several workers in the field earlier (see Fraenkel and Gunn 1961, for review) and, more recently, by Harris and Ringelberg in the studies mentioned above. Ringelberg (1964) also showed that when a daphnid in his apparatus was subjected to a rotating contrast, the animal attempted to follow the contrast. We have confirmed this observation (unpublished observations). Within the tracking zone, eye flick/fixation and tracking are simply the responses of the oculomotor system to stationary and moving stimuli, respectively (Fig. 9). Outside the tracking zone, flick and fixation perhaps serve to initiate an eye movement toward the stimulus. In this case the combination of eye and body movements would be necessary to bring the stimulus into the tracking zone where eye movement alone could follow it. Ringelberg et al. (1975) and Young (1981) have further proposed that it is the prominent contrast boundary provided by Snell's window which is the major orientational landmark for Daphnia. From the results of the experiments presented in this report, two possible visual goals for the oculomotor system may be proposed. The first is that the eye and body system may always attempt to point the null region at a strong contrast boundary. The second possibility is that the two contrast boundaries on either side of

Snell's window may provide a dual stimulus, and these cause the eye to remain stationary when their effects on the oculomotor system are balanced. A common orientation assumed by a daphnid during the day is with the body's long axis almost vertical and tilted slightly forward. In such a posture the animal continuously makes its 'hop and sink' (Stearns 1975) swimming movements which cause it to rock back and forth. The oculomotor system seems ideally suited to compensate for these motions and keep the eye properly pointed at (perhaps) the edge of Snell's window. A decisive experiment to support the aforementioned idea would be to record a free swimming daphnid with an optical system which gives a high enough magnification to identify the eye axis and a wide enough field of view to determine body orientation. The predicted results of such an experiment are that the eye and body would be seen to make complementary rotations, and that the eye would remain stationary relative to some landmark in the external world. Acknowledgements. We thank Jeff Camhi and Ken Smith for their comments on the manuscript. This work was supported by NIH Grant NS-14946.

References Collett TS, Land MF (1975) Visual control of flight behavior in the hoverfly Syritta pipiens. J Comp Physiol 99 : 1-66 Consi TR, Macagno ER (1985) The spectral sensitivity of eye movements in response to light flashes in Daphnia magna. J Comp Physiol A 156:135 143 Consi TR, Macagno ER, Necles N (1987) The oculomotor system of Daphnia magna. The eye muscles and their motor neurons. Cell Tissue Res 247:515 523 Consi TR, Passani MB, Macagno ER (1987) The two-light experiment in Daphnia magna Soc Neurosc Abstr 13 : 137 Flaster MS, Macagno ER, Schehr RS (1982) Mechanisms for the formation of synaptic connections in the isogenic nervous system of Daphnia magna. In: Spitzer NC (ed) Neuronal development. Plenum Press, New York, pp 267-296 Fraenkel G, Gunn DL (1961) The orientation of animals. Kineses, taxes and compass reactions. Dover, New York Frost BJ (1975) Eye movements in Daphnia pulex (De Geer). J Exp Biol 62:175-187 Harris JE, Mason P (1956) Vertical migration of eyeless Daphnia. Proc R Soc Lond B 145:280-290 Harris JE, Wolfe U K (1955) A laboratory study of vertical migration. Proc R Soc Lond B 144:329-354 Lythgoe JN (1979) The ecology of vision. Clarendon Press, Oxford Macagno ER, Lopresti V, Levinthal C (1973) Structure and development of neuronal connections in isogenic organisms: variations and similarities in the optic system of Daphnia magna. Proc Natl Acad Sci USA 70:57-61 Nalbach HO, Nalbach G (1987) Distribution of optokinetic sensitivity over the eye of crabs: its relation to habitat and possible role in the flow field analysis. J Comp Physiol A 160:127-135 Nilsson DE, Odselius R (1983) Regionally different optical systems in the compound eye of the water-flea Polyphemus (Cladocera, Crustacea). Proc R Soc Lond B 217:163-175 Odselius R, Nilsson DE (1983) Regionally different ommatidial structures in the compound eye of the water-flea Polyphemus (Cladocera, Crustacea). Proc R Soc Lond B 217:177-189

420 Ringelberg J (1964) The positively phototactic reaction of Daphnia magna Strauss. Neth J Sea Res 2:319-406 Ringelberg J, Flick BJG, Buis RC (1975) Contrast orientation in Daphnia magna and its significance for vertical plane orientation in the pelagic biotope in general. Neth J Zool 25: 454-475 R6hlich P, T6r6 I (1965) Fine structure of the compound eye of Daphnia in normal, dark- and strongly light-adapted state. In: Rohen JW (ed) The structure of the compound eye. II. Symposium. Schattauer, Stuttgart, pp 175-186 Schehr RS (1984) Spectral sensitivities of anatomically identified photoreceptors in the compound eye of Daphnia magna. PhD Dissertation, Columbia University Shaw SR, Stowe S (1982) Photoreception. In: Atwood HL, Sandeman DC (eds) The biology of Crustacea III. Neurobiology: structure and function. Academic Press, New York, pp 291367

Th. R. Consi et al. : Eye movements in Daphnia magna Sims SJ, Macagno ER (1985) Computer reconstruction of all the neurons in the optic ganglion of Daphnia magna. J Comp Neurol 233 : 12-29 Steams SC (1975) Light responses of Daphnia pulex. Limnol Oceanogr 20: 564-570 Wehner R (1981) Spatial vision in arthropods. In: Autrum H (ed) Vision in invertebrates (Handbook of sensory physiology vol. VII/6C). Springer, Berlin Heidelberg New York, pp 287-616 Young S (1981) Behavioural correlates in photoreception in Daphnia. In: Laverack MS, Cosens DJ (eds) Sense organs. Blackie, Glasgow, pp 49-63 Young S (1988) Chasing with a model eye. J Exp Biol 137:399-410 Young S, Downing AC (1976) The receptive fields of Daphnia ommatidia. J Exp Biol 64:185-202 Young S, Taylor VA (1988) Visually guided chases in Polyphemus pediculus. J Exp Biol 137:387-398