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Influence of exposure in ovo to different light wavelengths on the lateralization of social response in zebrafish larvae
Physiology & Behavior 157 (2016) 258–264
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Influence of exposure in ovo to different light wavelengths on the lateralization of social response in zebrafish larvae Valeria Anna Sovrano a,b,⁎, Cristiano Bertolucci c, Elena Frigato c, Augusto Foà c, Lesley J. Rogers d a
Center for Mind/Brain Sciences (CIMeC), University of Trento, Piazza Manifattura 1, Borgo Sacco, 38068 Rovereto (Trento), Italy Department of Psychology and Cognitive Science, University of Trento, C.so Bettini 31, 38068 Rovereto (Trento), Italy Department of Life Sciences and Biotechnology, University of Ferrara, Via Luigi Borsari 46, 44121 Ferrara, Italy d Centre for Neuroscience and Animal Behaviour, School of Science and Technology, University of New England, Armidale, NSW 2351, Australia b c
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Zebrafish larvae have preferential use of left eye to look at image in the mirror. • This behavioural asymmetry is shown only by larvae reared under a light/dark cycle. • Development in darkness or monochromatic light prevents this behavioural response.
a r t i c l e
i n f o
Article history: Received 1 December 2015 Received in revised form 27 January 2016 Accepted 10 February 2016 Available online 12 February 2016 Keywords: Zebrafish larvae Development Lateralisation Light exposure Eye use Wavelengths
a b s t r a c t Exposure of the chick embryo to different wavelengths of light of the same intensity has shown that only certain wavelengths may be important in generating visual asymmetries. This study aimed to detect the possible influence of different wavelengths of light on development of asymmetry of social recognition in zebrafish larvae, tested using the fish's mirror image as the stimulus. From fertilization until day 10 post-hatching zebrafish were kept in five different lighting conditions: natural light/dark (LD) cycle, complete darkness (DD), and artificial LD cycles with 14 h of monochromatic light (red, green, or violet light) and 10 h of darkness (rLD 14:10, gLD 14:10, vLD 14:10, respectively). On day 10 after hatching, the zebrafish larvae were subjected to a mirror test. A preference for using the left eye to scrutinize their mirror image was apparent only in zebrafish larvae exposed to and reared under a natural LD cycle, and not following exposure to any of other lighting conditions. These results are discussed with reference to other evidence of brain lateralization. © 2016 Elsevier Inc. All rights reserved.
1. Introduction ⁎ Corresponding author at: Center for Mind/Brain Sciences, University of Trento, Piazza Manifattura 1, Borgo Sacco, 38068 Rovereto (Trento), Italy. E-mail address: [email protected] (V.A. Sovrano).
http://dx.doi.org/10.1016/j.physbeh.2016.02.016 0031-9384/© 2016 Elsevier Inc. All rights reserved.
Exposure of embryos to light during critical stages of development has been shown to trigger development of lateralized brain function
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in the visual modality. This was demonstrated first in domestic chicks [1], then in pigeons [2] and later in zebrafish [3]. Exposure of chick embryos to white light during the final days of incubation leads to right eye, and left hemisphere, superiority in performance of a task in which grains of mash are discriminated from pebbles, known as the pebblefloor task, and to the control of attack and copulation responses by the left eye and right hemisphere [1, 4]. The same exposure to light also establishes left-eye advantage in detecting predators [5, 6]. These asymmetries are not present in chicks hatched from eggs incubated in the dark. They rely on the asymmetrical posture of the embryo in the egg, during the final days of incubation, such that the right eye only is stimulated by light after it passes through the shell and membranes [7]. Similarly, light exposure of pigeon embryos leads to right eye advantage in discriminating grains from grit [2] and in visuomotor speed of pecking [8]. Hence, light exposure during embryonic development is critical for the development of certain lateralized visual functions, even though some aspects of visual behaviour are present in darkincubated chicks: for example, specialization of the chick's left eye and right hemisphere to make choices to approach a familiar versus an unfamiliar conspecific does not rely on light exposure [9]. Imprinting also occurs in a lateralized manner in dark incubated chicks but light exposure does alter the lateralized recall of imprinting memory [10]. Recently, it has been shown that light exposure of chick embryos during early development from day 1 to day 4 of incubation influences the development of another kind of lateralized behaviour: in this case the light exposure effects a spatial bias to peck more at grains on the left side than on the right side [11]. Although the same side bias is also generated in chicks exposed to light during the last 3 days of incubation, development of this particular asymmetry is distinct from the lateralization of pebble-grain discrimination or attack and copulation, which develops only after light exposure on the final 3 days of incubation. Hence, it cannot be caused by the same processes [12]. Whereas light-exposure of the late-stage embryo affects laterality of behaviour by triggering asymmetrical development of visual connections between the thalamus and forebrain (see below), early light exposure occurs well before the retina and visual pathways begin to develop. Hence, it might depend on activation of genes that determine left-right differences in some early developing neural structure [11]. In zebrafish exposure of the embryo to white light early in incubation (days 1 and 3) generates behavioural asymmetry in the hatched fry. Fry hatched from eggs exposed to a normal light-dark cycle show asymmetry of responding to stimuli presented on the left or right side: they avoid a model of a predator when it is presented on their left side but approach it when it is presented on their right side [3, 13]. Conversely, fry hatched from eggs incubated in the dark do not show this asymmetry: they avoid a model predator seen on their left or right side. As Budaev and Andrew suggest [3], this effect of light exposure might rely on left-right differences in gene activation and in timing of this event. Left-right differences in the timing of neural genesis in the diencephalon of the zebrafish brain are involved in the development of asymmetry in the habenular subnuclei and their connections [14]. This left-right asynchrony might underlie light-stimulated development of lateralized brain function. Light-dependent differentiation of neurons in the habenulae that trigger cyclic release of melatonin from the pineal may be involved [15]. In chicks, Rogers and Krebs [16] addressed the question of whether the asymmetry that develops after exposure to light during the final stages of incubation is wavelength-dependent, and they found that lateralized performance on the pebble-grain task was generated by exposure to white light and, to a lesser extent, by exposure to green light but not by exposure to red light. Since visual asymmetry in the chick is known to depend on asymmetry of the thalamofugual visual pathway [17, 18], effects of the different wavelengths of light on development of the thalamofugal projections were also examined [16]. White, red and green light were all effective in triggering the development of asymmetry in these neural projections. Since it is well known that
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neural activity is essential for development of neuron connections [19, 20], these results indicate that the development of neurons in the thalamofugal visual pathway are not colour-coded. However, the presumably wider connectivity involved in lateralization of performance on the pebble-floor task develops mainly when broad-spectrum (white) light stimulates the right eye and connecting pathways. This suggests involvement of colour-coded neurons outside the thalamofugal visual pathway [16]. Recognizing the likelihood that the effect of light on development of the zebrafish may depend on different subcellular events than the effect of light on late-stage chick embryos, we decided to investigate the effectiveness of different wavelengths of light on lateralized visual behaviour in the zebrafish. It seemed to us timely to investigate the effect of exposing zebrafish embryos to different wavelengths of light and to test their laterality in a simulated social interaction. The test allows scoring of left versus right eye use in responding to the zebrafish's image in a mirror. The fish is placed into an aquarium with mirrors as walls. The eye used to attend to the fish's own image is scored by filming from above and recording the angle of the fish to the mirror. Study of the laterality of the mirror social response is well documented in the literature [21–24]. It is interesting that left eye use (coordinated by the right hemisphere) seems to be maintained both to view the image in the mirror and to view a real conspecific, probably because the left bias may be part of a more general specialization for establishing the identity of familiar stimuli [26–29]. On the other hand, visual lateralization of social inspection by zebrafish larvae (in the presence of mirrors or real conspecifics) does not seem to follow the time-course of development of social behaviour in this same species. The left bias does not change in young zebrafish tested at different ages (8, 12, 14, 21, 26 days), whereas social preference (measured in terms of a time spent close to a group of conspecifics) develops gradually and is robust in 3-week-old zebrafish (compare [27] with [30]). The laterality of left eye use to scrutinize social stimuli seems to be an early feature of social behaviour, supported by an asymmetric neural organization, which might undergo modifications in response to inputs from external stimuli, such as the environmental light. For this reason, we decided to investigate whether zebrafish that have been exposed to the natural cycle of sunlight and darkness during embryonic development exhibit a preference to view their image using the left eye (a general preference widespread in several vertebrates, [21, 22, 24–27, 29]), whereas this eye preference may not be present after rearing in the dark. We decided also to investigate whether red, green or violet light might be as effective as white light in triggering development of this asymmetry. 2. Material and methods 2.1. Zebrafish rearing Wild-type adult zebrafish were housed in 30 l glass aquaria (1 fish l−1) according to a standard method [31]. Fish were fed two times per day during daylight with dry food (TetraMin, Tetra GmbH, Germany). The water temperature was kept constant at 27 °C by thermostats (50 W, Juwel GmbH, Germany). Aquaria were placed in a lighted room (through the windows) and they were exposed to the natural photoperiod. For spontaneous spawning, 12 mature fish (6 male and 6 females) were transferred into a breeding aquarium during the afternoon. Spawning took place the next morning approximately 2 h after sunrise. Immediately after fertilization, eggs were pooled and distributed in 5 small glass breeding aquaria (N = 45 per aquarium) filled with 1.5 l of E3 embryo medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4) and maintained at the temperature of 27 °C under the experimental lighting conditions. Larvae were fed from day 6 post-fertilization (dpf) with Paramecium, Artemia larvae and dry food (TetraMin baby, Tetra GmbH, Germany). Since all aquaria were at the same temperature, all eggs hatched at about the same time (after
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3 days) and then they of the same age during the test. The experimental lighting conditions do not influence either survival rate or growth [32]. The body lengths of the zebrafish larvae were not statistically different at the day of testing (mean ± SD, mm; LD: 2.17 ± 0.68; DD: 1.93 ± 0.7; rLD: 2.2 ± 0.88; gLD: 1.73 ± 0.46; vLD: 1.94 ± 0.43; F(4,48) = 1.72, p N 0.16, ANOVA). 2.2. Experimental protocol Each breeding aquarium was exposed to a different lighting condition. In the first aquarium eggs/larvae were exposed to the natural light-dark (LD) cycle, the same conditions as during the reproductive phase. A second aquarium was covered by black plastic foil and kept in complete darkness in a dark room. The remaining 3 aquaria were exposed to 3 different wavelengths of light: 14:10 LD cycle with equalized photon flux density (≈ 1017 photons m−2 s− 1 nm− 1, spectrometer FieldSpec ASD, Colorado, USA), but different spectra. The exposures were long (peak at 657 nm), medium (peak at 530 nm) and short (peak at 410 nm) wavelengths. For the sake of brevity, throughout the rest of the paper we have called these conditions red (rLD), green (gLD), and violet (vLD), respectively. The choice, in the artificial lighting conditions, of a 14:10 LD cycle depends on the fact that these are the canonical conditions for breeding and experiments published previously used this cycle [27]. Moreover, the exposure to the natural photoperiod could offer a further guarantee of the effectiveness of such exposure to light, as already demonstrated in the artificial 14:10 LD cycle [27]. Illumination was provided by means of LED light lamps (18 LEDs per lamp; Superlight Technology Co. Ltd., China). The upper part of the tank was covered by a transparent, removable glass plate, on which rested a plastic transparent container, with the lamp. The container diffused the light uniformly inside the aquarium. These exposure conditions continued until testing. 2.3. Apparatus The apparatus (mirror test) was located in a darkened room and was identical to the one used in Sovrano and Andrew [27]; it consisted of a test tank (20 × 5 × 8 cm) with two mirrors as the two longer walls, and the two shorter walls were non-reflective white (Fig. 1a); The apparatus was placed inside a larger plastic rectangular tank (31 × 50 × 14.5 cm), filled with water and with two aquarium thermostats (Tethaht, Tetra GmbH, Germany), so that the external water heat maintained the internal water at a constant temperature of 27 °C. The apparatus was filled with the medium E3 up to a depth of 4 cm to maintain the same environment present during development of embryos. On the long sides of the tank were two daylight fluorescent tubes (each one 18 W, giving a total of 36 W, Lumilux T5, Osram GmbH, Germany) with black plastic covers, in order to avoid the dispersion of light upwards and ensure a better quality of video-recording. External to the apparatus there was gravel in order to stabilize the position of the apparatus and to prevent excessive upwards light reflection. A video camera (GZ-MG21E, JVC, Japan) was positioned above the apparatus in a central position on a tripod. 2.4. Procedure Subjects were 53 fry 10 dpf old (N = 12 in LD, N = 11 in DD, N = 10 in rLD, N = 11 in gLD, N = 9 in vLD). Fry were selected on the basis of their good quality of motion and the absence of external morphological malformations, which could create an impairment of the movement. For ethical reasons, we used the minimum number of animals compatible with having enough statistical power of the tests. On the test day (2 h before), in the same small glass breeding aquaria, in which eggs had been pooled and distributed there were now living fry. These were gently placed in the larger tank, in order to maintain the same breeding condition and to reduce to a minimum
Fig. 1. (A) Photography from above the apparatus used in the experiment, with two mirrors along the longest sides. (B) Schematic representation of the test apparatus, showing the position of the mirrors and the angles of viewing that define monocular vision with the right or left eye. Data were discarded when the fish was perpendicular to the mirror (binocular stimulation), when it formed an angle larger than 90° with respect to the closer mirror and when it was attached on the mirror for more than 6 s.
the stress for animals. Each fry was placed in turn in the middle of the apparatus in a dark cylinder (4 cm diameter) open at both ends, for a period of 2 min of acclimatization. After the cylinder removal, the behaviour of fry was video-recorded from above for 10 min. Fry positions were scored every 2 s by superimposition on the computer screen of a cursor on the long axis of the body, using the video recording. Observers did not know the lighting experimental conditions of any of the fry in videos and an inter-subjective validation criterion was randomly applied in different sequences of movement. Body angle was taken in relation to the closer mirror. We discarded positions when the fry was in a central strip 4 mm wide (Fig. 1b), when the fry was exactly perpendicular to the mirror (binocular stimulation), when it formed an angle larger than 180° with respect to the closer mirror and when the fry was touching the mirror, with its head and/or body, repeatedly showing escape behaviour (pushing against the mirror back and forth along the mirror, with characteristic movements of the tail) for more than 6 s (three observations). Positions in which fry were aligned parallel with the mirror (“parallel observations”: all those observations in which the fish had the body perfectly parallel - 180°, head aligned with the axis of the body- to the nearest mirror) and those in which fry were instead at an angle to the mirror (“angled observations”: all values of the body angle between 1°–179° towards the left–right eye use — and towards the right–left eye use) were recorded separately [21, 22, 27] (see Fig. 1b). The procedure was exactly the same as used in the mirror test by Sovrano and Andrew [27], in order to compare and to discuss both sets of experimental data. Eye use during the first and second 5 min of test was analyzed separately, since patterns commonly change over this period of time, as found in tests with adult teleost fish [21]. This proved to be the case here as well. An index of eye use was calculated as [(frequency of left eye use)/ (frequency of right eye use + frequency of left eye use)] × 100. Values significantly higher than 50% would thus indicate preference for left eye use, and values significantly lower than 50%, preference for right eye use. Significant departures from chance level (50%) were estimated by two-tailed one-sample t-test. Further analyses were carried out by analysis of variance (ANOVA). Data were normally distributed (p b 0.05, Kolmogorov–Smirnov's test).
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2.5. Ethics statement The present research was carried out in the laboratory of the University of Ferrara (Italy). All husbandry and experimental procedures complied with European Legislation for the Protection of Animals used for Scientific Purposes (Directive 2010/63/EU). The experimental protocol was previously authorized by the University of Ferrara Institutional Animal Care and Use Committee and the Italian Ministry of Health. 3. Results The results of five experimental conditions are shown in Figs. 2-3. The data were analyzed using analysis of variance (ANOVA), taking into consideration the condition of illumination as a between subjects factor and time as a within-subjects factor, to probe any differences in behaviour between the first 5 and second 5 min of test. The analyses were performed considering the angular observations, the parallel positions and the overall total observations (angular + parallel). In the case of angular observations, the ANOVA showed only a main statistical effect of the variable Lighting Condition (F(4,48) = 4.15, p = 0.006). There were no other statistically significant effects (time: F(1,48) = 0.01, p = 0.974; lighting condition × time: F(4,48) = 0.36, p = 0.834). In the case of parallel observations, the ANOVA revealed a statistically significant effect of the Lighting Condition (F(4,48) = 3.47, p = 0.014). Also in this case there were no other significant effects (Time: F(1,48) = 2.69, p = 0.108, Time × Lighting Condition: F(4,48) = 0.01, p = 0.060). For the overall observations (angular + parallel), there was a main effect of Lighting Condition (F(4,48) = 4.89, p = 0.002), whereas the other variables were not significant (time: F(1,48) = 0.37, p = 0.548; Lighting Condition × Time: F(4,48) = 0.02, p = 0.435). Since there was no effect of Time, an ANOVA was conducted on the total observations (angular + parallel in the first 5 min and in the second 5 min of test). The analysis of variance (ANOVA) showed a significant effect of Lighting Condition (F(4,48) = 5, p = 0.002). In Fig. 2 we compared the overall index (angular + parallel). From the graph it is apparent that the significance of the variable lighting condition was attributable to the strongly lateralized behaviour in the use of the left eye from fish reared in the LD condition. Note that the strength of this lateralized behaviour emerged also considering total observations only in the first 5 min of test (Lighting Condition: F(4,48) = 2.76, p = 0.038). Because the ANOVA showed only the presence of the main effect of Lighting Condition, we were allowed to apply a one-sample t-test to
Fig. 3. Graph showing, in the natural LD cycle (A) and DD (B) condition, the percentage of the left eye use (group means with S.E.M.), considering separately indexes of different positions in front of the mirror (angular, parallel and angular + parallel) in the first 5 min (AI 5, PI 5 I 5) and in the second 5 min (AI 10, PI 10, I 10) of test.
evaluate further, in each of the Lighting Conditions (LD — Fig. 3A, DD — Fig. 3B, rLD — Fig. 4A, gLD — Fig. 4B, vLD — Fig. 4C). Such analysis were applied to the deviation of laterality index from the random value (50%) for each of the dependent variables examined: index of angular position in the first 5 min (ang_index_5 or AI 5), index of parallel position in the first 5 min (par_index_5 or PI 5), total index (angular + parallel positions) in the first 5 min (index_5 or I 5), index of angular position in the second 5 min (ang_index_10 or AI 10), index of parallel position in the second 5 min (par_index_10 or PI 10), total index (angular + parallel positions) in the second 5 min (index_10 or I 10), global index (angular + parallel positions) in all 10 min of test (tot_index or Tot I). The results are summarized in the Table 1 and in Figs. 3 and 4. Results indicate the preference of the use of the left eye to look at the image in the mirror for fish reared in the natural LD condition (Fig. 3). By contrast, in all other Lighting Conditions there was no statistically significant value and therefore no expression of laterality in looking at the image in the mirror (Fig. 4). 4. Discussion
Fig. 2. Graph showing the percentage of the left eye use in different lighting conditions (group means with S.E.M.): LD = natural light/dark cycle, DD = continuous darkness, rLD = 14:10 red light-dark cycle, gLD = 14:10 green light-dark cycle, vLD = 14:10 violet light-dark cycle.
The lateralization (left eye use) that we measured on day 10 after hatching was evident only in those zebrafish that were exposed to natural light/dark conditions. Previously it had been shown [27] that a cycle
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Fig. 4. Presented here are the results of fry raised in the 14:10 LD cycles with monochromatic light of different wavelengths: (A) red, (B) green, and (C) violet light (rLD, gLD, vLD, respectively). The percentage of the left eye use is presented as group means with S.E.M., as in Fig. 3.
of 14 h of artificial white light and 10 h of darkness has, in the zebrafish larvae, the same effect of establishing lateralization of mirror-image viewing as we found here for the natural light/dark cycle (LD about 12:12). Keeping the developing embryos and the early post-hatching
zebrafish in darkness prevents the development of this asymmetry. Hence, lateralization of response by zebrafish manifested by seeing their image in a mirror requires exposure to white light in ovo and during early post-hatching development. After exposure to a cycle of 14 h of red, green or violet light and 10 h of darkness, no laterality of mirror-image viewing developed. It is, therefore, apparent that the neural events involved in development of this laterality require stimulation by a broad spectrum of light and that they do not develop after exposure to specific wavelengths alone. Certainly, red light alone, green light alone or violet light alone does not enable the development of this particular laterality. Since we controlled for the light intensity, we can be sure that a broad-spectrum of wavelengths is required, indicating that colour-coded neurons are involved. This result is quite similar to that found for the development of behavioural lateralization in chicks [16]. As mentioned in the Introduction, the lateralization in chicks develops after exposure of the embryo to white light, and to a lesser extent after exposure to green light, but not after exposure to red light (violet was not tested). It is interesting to note that the behavioural lateralization in both zebrafish and chicks differs from the demonstrated light-dependent asymmetry in the thalamofugal visual system of the chick. These neurons grow asymmetrically after stimulation by white, red or green light [16]. Presumably this mismatch between neuronal and behavioural response to light exposure depends on extra-visual neurons being used in control of the behaviour or in use of another visual pathway the development of which is colour-coded. In birds this could be the tectofugal visual system, which is known to respond to colour, whereas the thalamofugal visual system does not [33, 34]. However, Deng and Rogers [18] have shown that only the thalamofugal-recipient region of the chick forebrain is essential in performance of the pebble-grain task, and not the forebrain region that receives input from the tectofugal visual system. Hence, it seems that development colour-coded neurons outside the afferent visual input pathways to the forebrain are involved. This may be the case also in zebrafish. The zebrafish eye achieves its emmetropic form, typical of the adult, by as early as 72 h post-fertilization [35]. Prior to this, cone-visual ability has developed: cones develop by 40–50 h post-fertilization, bipolar cells by 60 h post-fertilization and both amacrine and horizontal cells by 50 h post-fertilization. These stages of development fall well within the period of exposure to different wavelengths of light that we used and so could potentially have been affected by the different treatments. By contrast, since retinal rod cells do not develop a functional role in vision until 15–40 dpf [36], much of their development occurs after the period of exposure used by us (our period of exposure was from fertilization to day 10 post-fertilization). Although very early stages of rod-cell differentiation could, perhaps, have been affected by exposure to different wavelengths of light, this seems less probable than an effect on cone cells. The tetrachromatic colour vision of the zebrafish depends on four types of cones [36]: viz., long-wavelength cones (L cones) with peak sensitivity in the red region of the spectrum (556–564 nm), M cones with a peak sensitivity in the green region (473–480 nm), S cones in the blue region (407–417 nm and these would be stimulated by the violet light that we used), and UV cones with their peak sensitivity in the ultraviolet region (360–361 nm). Our results indicate that more than one, and likely all four, of these types of cones must be stimulated during early development if lateralized visual responding to a mirror image is to develop. Stimulation of any one type of cone alone was not sufficient to generate the behavioural laterality. We used cyclic exposure to the different wavelengths of light, which was important since Robinson and Dowling [37] have shown that rearing zebrafish in constant light or dark leads to deficits in visual behaviour, even though these effects cause no detectable cellular deficits in retinal anatomy. In fact, timing-dependent activity of the retinal ganglion cells is necessary for refined development of retinotectal projections
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Table 1 Summary of the results. Statistical significant differences are in bold.
LD DD rLD gLD vLD
AI 5
PI 5
I5
AI 10
PI 10
I 10
Tot I
t11 = 3.65 p = 0.004 t10 = 0.93 p = 0.376 t9 = −0.30 p = 0.768 t10 = 1.54 p = 0.154 t8 = 0.89 p = 0.401
t11 = 1.84 p = 0.093 t10 = 1.33 p = 0.214 t9 = 1.96 p = 0.082 t10 = −0.54 p = 0.601 t8 = 0.58 p = 0.576
t11 = 3.42 p = 0.006 t10 = 1.28 p = 0.228 t9 = −0.63 p = 0.542 t10 = 0.59 p = 0.568 t8 = 0.98 p = 0.356
t11 = 2.53 p = 0.028 t10 = 0.24 p = 0.813 t9 = −0.02 p = 0.986 t10 = 1.71 p = 0.117 t8 = −0.55 p = 0.595
t11 = 3.14 p = 0.009 t10 = 0.13 p = 0.902 t9 = −0.40 p = 0.696 t10 = 0.66 p = 0.526 t8 = 0.83 p = 0.428
t11 = 3.23 p = 0.008 t10 = 0.36 p = 0.723 t9 = −0.24 p = 0.816 t10 = 1.72 p = 0.116 t8 = −0.16 p = 0.875
t11 = 4.31 p = 0.001 t10 = 0.74 p = 0.478 t9 = −0.81 p = 0.437 t10 = 1.38 p = 0.196 t8 = 1.51 p = 0.170
[38] and cyclic short-wavelength stimulation is necessary for development of UV sensitivity [39]. Such wavelength specificity is not apparent in the behavioural asymmetry that we investigated. The question arising now is whether the white-light dependent development of the behavioural lateralization is generated by asymmetry of eye stimulation, as known to be so in the case of the late embryonic effect of light on lateralization in chicks, or by light-dependent asymmetries in the timing of gene expression during development [40]. The latter is more likely to be the underlying mechanism since zebrafish eggs are transparent and, unlike chick embryos, the orientation of the embryo's body is unlikely to cause lateralized light stimulation. 5. Conclusions Asymmetry of the eyed use to view their image in a mirror is a characteristic of zebrafish raised in the natural photoperiod and in laboratory conditions in which they are exposed to a cycle of white light and darkness close to the natural cycle. Since no asymmetry develops if the zebrafish eggs and newly hatched fry are raised in the dark or are exposed to cycling monochromatic light (red, green or violet), we conclude that broad-spectrum stimulation is necessary to generate the neural asymmetry expressed as side differences in visual aspects of social behaviour. Acknowledgements This study was partly supported by an ERC Grant (ERC-2011ADG_20110406, Project No: 295517, PREMESOR) and by the University of Ferrara (FAR 2014). We wish to thank Lorella Fiorino for her help with the experiments and Andrea Margutti for the technical assistance. References [1] L.J. Rogers, Light experience and asymmetry of brain function in chickens, Nature 297 (1982) 223–225. [2] O. Güntürkün, The ontogeny of visual lateralization in pigeons, Ger. J. Psychol. 17 (1993) 276–287. [3] S. Budaev, R.J. Andrew, Shyness and behavioural asymmetries in larval zebrafish (Brachydanio rerio) incubated in the dark, Behavior 146 (2009) 1037–1052. [4] J.V. Zappia, L.J. Rogers, Light experience during development affects asymmetry of forebrain function in chickens, Brain Res. 313 (1983) 93–106. [5] L.J. Rogers, Evolution of hemispheric specialization: advantages and disadvantages, Brain Lang. 73 (2000) 236–253. [6] L.J. Rogers, Development and function of lateralization in the avian brain, Brain Res. Bull. 76 (2008) 235–244. [7] L.J. Rogers, Light input and the reversal of functional lateralization in the chicken brain, Behav. Brain Res. 38 (1990) 211–221. [8] M. Skiba, B. Diekamp, O. Güntürkün, Embryonic light stimulation induces different asymmetries in visuoperceptual and visuomotor pathways of pigeons, Behav. Brain Res. 134 (2002) 149–156. [9] R.J. Andrew, A.N. Johnston, A. Robins, L.J. Rogers, Light experience and the development of behavioural lateralisation in chicks. II. Choice of familiar versus unfamiliar model social partner, Behav. Brain Res. 5 (155) (2004) 67–76.
[10] A.N. Johnston, L.J. Rogers, Light exposure of chick embryo influences lateralized recall of imprinting memory, Behav. Neurosci. 113 (1999) 1267–1273. [11] C. Chiandetti, J. Gallusi, R.J. Andrew, G. Vallortigara, Early-light embryonic stimulation suggests a second route, via gene activation, to cerebral lateralization in vertebrates, Sci. Report. 3 (2013) 2701. [12] L.J. Rogers, Asymmetry of brain and behavior in animals: its development, function, and human relevance, Genesis 52 (2014) 555–571. [13] S. Budaev, R.J. Andrew, Patterns of early embryonic light exposure determine behavioural asymmetries in zebrafish: a habenular hypothesis, Behav. Brain Res. 200 (2009) 91–94. [14] H. Aizawa, M. Goto, T. Sato, H. Okamoto, Temporally regulated asymmetric neurogenesis causes left-right difference in the zebrafish habenular structures, Dev. Cell 12 (2007) 87–98. [15] N.H. de Borsetti, B.J. Dean, E.J. Bain, J.A. Clanton, R.W. Taylor, J.T. Gamse, Light and melatonin schedule neuronal differentiation in the habenular nuclei, Dev. Biol. 358 (2011) 251–261. [16] L.J. Rogers, G.A. Krebs, Exposure to different wavelengths of light and the development of structural and functional asymmetries in the chicken, Behav. Brain Res. 80 (1996) 65–73. [17] L.J. Rogers, H.S. Sink, Transient asymmetry in the projections of the rostral thalamus to the visual hyperstriatum of the chicken, and reversal of its direction by light exposure, Exp. Brain Res. 70 (1988) 378–384. [18] C. Deng, L.J. Rogers, Differential contributions of the two visual pathways to functional lateralization in chicks, Behav. Brain Res. 87 (1997) 173–182. [19] N.C. Spitzer, Electrical activity in early neuronal development, Nature 444 (2006) 707–712. [20] N.V. De Marco García, T. Karayannis, G. Fishell, Neuronal activity is required for the development of specific cortical interneuron subtypes, Nature 472 (2011) 351–355. [21] V.A. Sovrano, C. Rainoldi, A. Bisazza, G. Vallortigara, Roots of brain specializations: preferential left-eye use during mirror- image inspection in six species of teleost fish, Behav. Brain Res. 106 (1999) 175–180. [22] V.A. Sovrano, A. Bisazza, G. Vallortigara, Lateralization of response to social stimuli in fishes: a comparison between different methods and species, Physiol. Behav. 74 (2001) 237–244. [23] A. De Santi, V.A. Sovrano, A. Bisazza, G. Vallortigara, Mosquitofish display differential left- and right-eye use during mirror image scrutiny and predator inspection responses, Anim. Behav. 61 (2001) 305–310. [24] A. Bisazza, A. De Santi, S. Bonso, V.A. Sovrano, Frogs and toads in front of a mirror: Lateralisation of response to social stimuli in tadpoles of five anuran species, Behav. Brain Res. 134 (2002) 417–424. [25] A. Valenti, V.A. Sovrano, P. Zucca, G. Vallortigara, Visual lateralisation in quails (Coturnix coturnix), Laterality 8 (2003) 67–78. [26] V.A. Sovrano, Visual lateralization in response to familiar and unfamiliar stimuli in fish, Behav. Brain Res. 152 (2004) 385–391. [27] V.A. Sovrano, R.J. Andrew, Eye use during viewing a reflection: behavioural lateralisation in zebrafish larvae, Behav. Brain Res. 167 (2006) 226–231. [28] M. Dadda, V.A. Sovrano, A. Bisazza, Temporal pattern of social aggregation in tadpoles and its influence on the measurement of lateralised response to social stimuli, Physiol. Behav. 78 (2003) 337–341. [29] P. Zucca, V.A. Sovrano, Animal lateralization and social recognition: quails use their left visual hemifield when approaching a companion and their right visual hemifield when approaching a stranger, Cortex 44 (2008) 13–20. [30] E. Dreosti, G. Lopes, A.R. Kampff, S.W. Wilson, Development of social behavior in young zebrafish, Front Neural Circuits 18 (9) (2015) 39. [31] C. Nüsslein-Volhard, D.T. Gilmour, R. Dahm, Zebrafish: a practical approach, Oxford University Press, New York, 2002. [32] V. Di Rosa, E. Frigato, J.F. López-Olmeda, F.J. Sánchez-Vázquez, C. Bertolucci, The light wavelength affects the ontogeny of clock gene expression and activity rhythms in zebrafish larvae, PLoS One 6 (2015), 10:e0132235. [33] W. Hodos, Color discrimination deficits after lesions of nucleus rotundus in pigeons, Brain Behav. Evol. 2 (1969) 185–200. [34] W. Hodos, The visual capabilities of birds, in: Zeigler HP, Bischof HJ (Eds.), Vision, Brain, and Behavior in Birds, MIT Press, Cambridge 1993, pp. 63–76. [35] J. Bilotta, S. Saszik, The zebrafish as a model visual system, Int. J. Dev. Neurosci. 19 (2001) 621–629. [36] V.C. Fleisch, S.C. Neuhauss, Visual behavior in zebrafish, Zebrafish 3 (2006) 191–201.
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Physiology & Behavior journal homepage: www.elsevier.com/locate/phb
Influence of exposure in ovo to different light wavelengths on the lateralization of social response in zebrafish larvae Valeria Anna Sovrano a,b,⁎, Cristiano Bertolucci c, Elena Frigato c, Augusto Foà c, Lesley J. Rogers d a
Center for Mind/Brain Sciences (CIMeC), University of Trento, Piazza Manifattura 1, Borgo Sacco, 38068 Rovereto (Trento), Italy Department of Psychology and Cognitive Science, University of Trento, C.so Bettini 31, 38068 Rovereto (Trento), Italy Department of Life Sciences and Biotechnology, University of Ferrara, Via Luigi Borsari 46, 44121 Ferrara, Italy d Centre for Neuroscience and Animal Behaviour, School of Science and Technology, University of New England, Armidale, NSW 2351, Australia b c
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Zebrafish larvae have preferential use of left eye to look at image in the mirror. • This behavioural asymmetry is shown only by larvae reared under a light/dark cycle. • Development in darkness or monochromatic light prevents this behavioural response.
a r t i c l e
i n f o
Article history: Received 1 December 2015 Received in revised form 27 January 2016 Accepted 10 February 2016 Available online 12 February 2016 Keywords: Zebrafish larvae Development Lateralisation Light exposure Eye use Wavelengths
a b s t r a c t Exposure of the chick embryo to different wavelengths of light of the same intensity has shown that only certain wavelengths may be important in generating visual asymmetries. This study aimed to detect the possible influence of different wavelengths of light on development of asymmetry of social recognition in zebrafish larvae, tested using the fish's mirror image as the stimulus. From fertilization until day 10 post-hatching zebrafish were kept in five different lighting conditions: natural light/dark (LD) cycle, complete darkness (DD), and artificial LD cycles with 14 h of monochromatic light (red, green, or violet light) and 10 h of darkness (rLD 14:10, gLD 14:10, vLD 14:10, respectively). On day 10 after hatching, the zebrafish larvae were subjected to a mirror test. A preference for using the left eye to scrutinize their mirror image was apparent only in zebrafish larvae exposed to and reared under a natural LD cycle, and not following exposure to any of other lighting conditions. These results are discussed with reference to other evidence of brain lateralization. © 2016 Elsevier Inc. All rights reserved.
1. Introduction ⁎ Corresponding author at: Center for Mind/Brain Sciences, University of Trento, Piazza Manifattura 1, Borgo Sacco, 38068 Rovereto (Trento), Italy. E-mail address: [email protected] (V.A. Sovrano).
http://dx.doi.org/10.1016/j.physbeh.2016.02.016 0031-9384/© 2016 Elsevier Inc. All rights reserved.
Exposure of embryos to light during critical stages of development has been shown to trigger development of lateralized brain function
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in the visual modality. This was demonstrated first in domestic chicks [1], then in pigeons [2] and later in zebrafish [3]. Exposure of chick embryos to white light during the final days of incubation leads to right eye, and left hemisphere, superiority in performance of a task in which grains of mash are discriminated from pebbles, known as the pebblefloor task, and to the control of attack and copulation responses by the left eye and right hemisphere [1, 4]. The same exposure to light also establishes left-eye advantage in detecting predators [5, 6]. These asymmetries are not present in chicks hatched from eggs incubated in the dark. They rely on the asymmetrical posture of the embryo in the egg, during the final days of incubation, such that the right eye only is stimulated by light after it passes through the shell and membranes [7]. Similarly, light exposure of pigeon embryos leads to right eye advantage in discriminating grains from grit [2] and in visuomotor speed of pecking [8]. Hence, light exposure during embryonic development is critical for the development of certain lateralized visual functions, even though some aspects of visual behaviour are present in darkincubated chicks: for example, specialization of the chick's left eye and right hemisphere to make choices to approach a familiar versus an unfamiliar conspecific does not rely on light exposure [9]. Imprinting also occurs in a lateralized manner in dark incubated chicks but light exposure does alter the lateralized recall of imprinting memory [10]. Recently, it has been shown that light exposure of chick embryos during early development from day 1 to day 4 of incubation influences the development of another kind of lateralized behaviour: in this case the light exposure effects a spatial bias to peck more at grains on the left side than on the right side [11]. Although the same side bias is also generated in chicks exposed to light during the last 3 days of incubation, development of this particular asymmetry is distinct from the lateralization of pebble-grain discrimination or attack and copulation, which develops only after light exposure on the final 3 days of incubation. Hence, it cannot be caused by the same processes [12]. Whereas light-exposure of the late-stage embryo affects laterality of behaviour by triggering asymmetrical development of visual connections between the thalamus and forebrain (see below), early light exposure occurs well before the retina and visual pathways begin to develop. Hence, it might depend on activation of genes that determine left-right differences in some early developing neural structure [11]. In zebrafish exposure of the embryo to white light early in incubation (days 1 and 3) generates behavioural asymmetry in the hatched fry. Fry hatched from eggs exposed to a normal light-dark cycle show asymmetry of responding to stimuli presented on the left or right side: they avoid a model of a predator when it is presented on their left side but approach it when it is presented on their right side [3, 13]. Conversely, fry hatched from eggs incubated in the dark do not show this asymmetry: they avoid a model predator seen on their left or right side. As Budaev and Andrew suggest [3], this effect of light exposure might rely on left-right differences in gene activation and in timing of this event. Left-right differences in the timing of neural genesis in the diencephalon of the zebrafish brain are involved in the development of asymmetry in the habenular subnuclei and their connections [14]. This left-right asynchrony might underlie light-stimulated development of lateralized brain function. Light-dependent differentiation of neurons in the habenulae that trigger cyclic release of melatonin from the pineal may be involved [15]. In chicks, Rogers and Krebs [16] addressed the question of whether the asymmetry that develops after exposure to light during the final stages of incubation is wavelength-dependent, and they found that lateralized performance on the pebble-grain task was generated by exposure to white light and, to a lesser extent, by exposure to green light but not by exposure to red light. Since visual asymmetry in the chick is known to depend on asymmetry of the thalamofugual visual pathway [17, 18], effects of the different wavelengths of light on development of the thalamofugal projections were also examined [16]. White, red and green light were all effective in triggering the development of asymmetry in these neural projections. Since it is well known that
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neural activity is essential for development of neuron connections [19, 20], these results indicate that the development of neurons in the thalamofugal visual pathway are not colour-coded. However, the presumably wider connectivity involved in lateralization of performance on the pebble-floor task develops mainly when broad-spectrum (white) light stimulates the right eye and connecting pathways. This suggests involvement of colour-coded neurons outside the thalamofugal visual pathway [16]. Recognizing the likelihood that the effect of light on development of the zebrafish may depend on different subcellular events than the effect of light on late-stage chick embryos, we decided to investigate the effectiveness of different wavelengths of light on lateralized visual behaviour in the zebrafish. It seemed to us timely to investigate the effect of exposing zebrafish embryos to different wavelengths of light and to test their laterality in a simulated social interaction. The test allows scoring of left versus right eye use in responding to the zebrafish's image in a mirror. The fish is placed into an aquarium with mirrors as walls. The eye used to attend to the fish's own image is scored by filming from above and recording the angle of the fish to the mirror. Study of the laterality of the mirror social response is well documented in the literature [21–24]. It is interesting that left eye use (coordinated by the right hemisphere) seems to be maintained both to view the image in the mirror and to view a real conspecific, probably because the left bias may be part of a more general specialization for establishing the identity of familiar stimuli [26–29]. On the other hand, visual lateralization of social inspection by zebrafish larvae (in the presence of mirrors or real conspecifics) does not seem to follow the time-course of development of social behaviour in this same species. The left bias does not change in young zebrafish tested at different ages (8, 12, 14, 21, 26 days), whereas social preference (measured in terms of a time spent close to a group of conspecifics) develops gradually and is robust in 3-week-old zebrafish (compare [27] with [30]). The laterality of left eye use to scrutinize social stimuli seems to be an early feature of social behaviour, supported by an asymmetric neural organization, which might undergo modifications in response to inputs from external stimuli, such as the environmental light. For this reason, we decided to investigate whether zebrafish that have been exposed to the natural cycle of sunlight and darkness during embryonic development exhibit a preference to view their image using the left eye (a general preference widespread in several vertebrates, [21, 22, 24–27, 29]), whereas this eye preference may not be present after rearing in the dark. We decided also to investigate whether red, green or violet light might be as effective as white light in triggering development of this asymmetry. 2. Material and methods 2.1. Zebrafish rearing Wild-type adult zebrafish were housed in 30 l glass aquaria (1 fish l−1) according to a standard method [31]. Fish were fed two times per day during daylight with dry food (TetraMin, Tetra GmbH, Germany). The water temperature was kept constant at 27 °C by thermostats (50 W, Juwel GmbH, Germany). Aquaria were placed in a lighted room (through the windows) and they were exposed to the natural photoperiod. For spontaneous spawning, 12 mature fish (6 male and 6 females) were transferred into a breeding aquarium during the afternoon. Spawning took place the next morning approximately 2 h after sunrise. Immediately after fertilization, eggs were pooled and distributed in 5 small glass breeding aquaria (N = 45 per aquarium) filled with 1.5 l of E3 embryo medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4) and maintained at the temperature of 27 °C under the experimental lighting conditions. Larvae were fed from day 6 post-fertilization (dpf) with Paramecium, Artemia larvae and dry food (TetraMin baby, Tetra GmbH, Germany). Since all aquaria were at the same temperature, all eggs hatched at about the same time (after
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3 days) and then they of the same age during the test. The experimental lighting conditions do not influence either survival rate or growth [32]. The body lengths of the zebrafish larvae were not statistically different at the day of testing (mean ± SD, mm; LD: 2.17 ± 0.68; DD: 1.93 ± 0.7; rLD: 2.2 ± 0.88; gLD: 1.73 ± 0.46; vLD: 1.94 ± 0.43; F(4,48) = 1.72, p N 0.16, ANOVA). 2.2. Experimental protocol Each breeding aquarium was exposed to a different lighting condition. In the first aquarium eggs/larvae were exposed to the natural light-dark (LD) cycle, the same conditions as during the reproductive phase. A second aquarium was covered by black plastic foil and kept in complete darkness in a dark room. The remaining 3 aquaria were exposed to 3 different wavelengths of light: 14:10 LD cycle with equalized photon flux density (≈ 1017 photons m−2 s− 1 nm− 1, spectrometer FieldSpec ASD, Colorado, USA), but different spectra. The exposures were long (peak at 657 nm), medium (peak at 530 nm) and short (peak at 410 nm) wavelengths. For the sake of brevity, throughout the rest of the paper we have called these conditions red (rLD), green (gLD), and violet (vLD), respectively. The choice, in the artificial lighting conditions, of a 14:10 LD cycle depends on the fact that these are the canonical conditions for breeding and experiments published previously used this cycle [27]. Moreover, the exposure to the natural photoperiod could offer a further guarantee of the effectiveness of such exposure to light, as already demonstrated in the artificial 14:10 LD cycle [27]. Illumination was provided by means of LED light lamps (18 LEDs per lamp; Superlight Technology Co. Ltd., China). The upper part of the tank was covered by a transparent, removable glass plate, on which rested a plastic transparent container, with the lamp. The container diffused the light uniformly inside the aquarium. These exposure conditions continued until testing. 2.3. Apparatus The apparatus (mirror test) was located in a darkened room and was identical to the one used in Sovrano and Andrew [27]; it consisted of a test tank (20 × 5 × 8 cm) with two mirrors as the two longer walls, and the two shorter walls were non-reflective white (Fig. 1a); The apparatus was placed inside a larger plastic rectangular tank (31 × 50 × 14.5 cm), filled with water and with two aquarium thermostats (Tethaht, Tetra GmbH, Germany), so that the external water heat maintained the internal water at a constant temperature of 27 °C. The apparatus was filled with the medium E3 up to a depth of 4 cm to maintain the same environment present during development of embryos. On the long sides of the tank were two daylight fluorescent tubes (each one 18 W, giving a total of 36 W, Lumilux T5, Osram GmbH, Germany) with black plastic covers, in order to avoid the dispersion of light upwards and ensure a better quality of video-recording. External to the apparatus there was gravel in order to stabilize the position of the apparatus and to prevent excessive upwards light reflection. A video camera (GZ-MG21E, JVC, Japan) was positioned above the apparatus in a central position on a tripod. 2.4. Procedure Subjects were 53 fry 10 dpf old (N = 12 in LD, N = 11 in DD, N = 10 in rLD, N = 11 in gLD, N = 9 in vLD). Fry were selected on the basis of their good quality of motion and the absence of external morphological malformations, which could create an impairment of the movement. For ethical reasons, we used the minimum number of animals compatible with having enough statistical power of the tests. On the test day (2 h before), in the same small glass breeding aquaria, in which eggs had been pooled and distributed there were now living fry. These were gently placed in the larger tank, in order to maintain the same breeding condition and to reduce to a minimum
Fig. 1. (A) Photography from above the apparatus used in the experiment, with two mirrors along the longest sides. (B) Schematic representation of the test apparatus, showing the position of the mirrors and the angles of viewing that define monocular vision with the right or left eye. Data were discarded when the fish was perpendicular to the mirror (binocular stimulation), when it formed an angle larger than 90° with respect to the closer mirror and when it was attached on the mirror for more than 6 s.
the stress for animals. Each fry was placed in turn in the middle of the apparatus in a dark cylinder (4 cm diameter) open at both ends, for a period of 2 min of acclimatization. After the cylinder removal, the behaviour of fry was video-recorded from above for 10 min. Fry positions were scored every 2 s by superimposition on the computer screen of a cursor on the long axis of the body, using the video recording. Observers did not know the lighting experimental conditions of any of the fry in videos and an inter-subjective validation criterion was randomly applied in different sequences of movement. Body angle was taken in relation to the closer mirror. We discarded positions when the fry was in a central strip 4 mm wide (Fig. 1b), when the fry was exactly perpendicular to the mirror (binocular stimulation), when it formed an angle larger than 180° with respect to the closer mirror and when the fry was touching the mirror, with its head and/or body, repeatedly showing escape behaviour (pushing against the mirror back and forth along the mirror, with characteristic movements of the tail) for more than 6 s (three observations). Positions in which fry were aligned parallel with the mirror (“parallel observations”: all those observations in which the fish had the body perfectly parallel - 180°, head aligned with the axis of the body- to the nearest mirror) and those in which fry were instead at an angle to the mirror (“angled observations”: all values of the body angle between 1°–179° towards the left–right eye use — and towards the right–left eye use) were recorded separately [21, 22, 27] (see Fig. 1b). The procedure was exactly the same as used in the mirror test by Sovrano and Andrew [27], in order to compare and to discuss both sets of experimental data. Eye use during the first and second 5 min of test was analyzed separately, since patterns commonly change over this period of time, as found in tests with adult teleost fish [21]. This proved to be the case here as well. An index of eye use was calculated as [(frequency of left eye use)/ (frequency of right eye use + frequency of left eye use)] × 100. Values significantly higher than 50% would thus indicate preference for left eye use, and values significantly lower than 50%, preference for right eye use. Significant departures from chance level (50%) were estimated by two-tailed one-sample t-test. Further analyses were carried out by analysis of variance (ANOVA). Data were normally distributed (p b 0.05, Kolmogorov–Smirnov's test).
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2.5. Ethics statement The present research was carried out in the laboratory of the University of Ferrara (Italy). All husbandry and experimental procedures complied with European Legislation for the Protection of Animals used for Scientific Purposes (Directive 2010/63/EU). The experimental protocol was previously authorized by the University of Ferrara Institutional Animal Care and Use Committee and the Italian Ministry of Health. 3. Results The results of five experimental conditions are shown in Figs. 2-3. The data were analyzed using analysis of variance (ANOVA), taking into consideration the condition of illumination as a between subjects factor and time as a within-subjects factor, to probe any differences in behaviour between the first 5 and second 5 min of test. The analyses were performed considering the angular observations, the parallel positions and the overall total observations (angular + parallel). In the case of angular observations, the ANOVA showed only a main statistical effect of the variable Lighting Condition (F(4,48) = 4.15, p = 0.006). There were no other statistically significant effects (time: F(1,48) = 0.01, p = 0.974; lighting condition × time: F(4,48) = 0.36, p = 0.834). In the case of parallel observations, the ANOVA revealed a statistically significant effect of the Lighting Condition (F(4,48) = 3.47, p = 0.014). Also in this case there were no other significant effects (Time: F(1,48) = 2.69, p = 0.108, Time × Lighting Condition: F(4,48) = 0.01, p = 0.060). For the overall observations (angular + parallel), there was a main effect of Lighting Condition (F(4,48) = 4.89, p = 0.002), whereas the other variables were not significant (time: F(1,48) = 0.37, p = 0.548; Lighting Condition × Time: F(4,48) = 0.02, p = 0.435). Since there was no effect of Time, an ANOVA was conducted on the total observations (angular + parallel in the first 5 min and in the second 5 min of test). The analysis of variance (ANOVA) showed a significant effect of Lighting Condition (F(4,48) = 5, p = 0.002). In Fig. 2 we compared the overall index (angular + parallel). From the graph it is apparent that the significance of the variable lighting condition was attributable to the strongly lateralized behaviour in the use of the left eye from fish reared in the LD condition. Note that the strength of this lateralized behaviour emerged also considering total observations only in the first 5 min of test (Lighting Condition: F(4,48) = 2.76, p = 0.038). Because the ANOVA showed only the presence of the main effect of Lighting Condition, we were allowed to apply a one-sample t-test to
Fig. 3. Graph showing, in the natural LD cycle (A) and DD (B) condition, the percentage of the left eye use (group means with S.E.M.), considering separately indexes of different positions in front of the mirror (angular, parallel and angular + parallel) in the first 5 min (AI 5, PI 5 I 5) and in the second 5 min (AI 10, PI 10, I 10) of test.
evaluate further, in each of the Lighting Conditions (LD — Fig. 3A, DD — Fig. 3B, rLD — Fig. 4A, gLD — Fig. 4B, vLD — Fig. 4C). Such analysis were applied to the deviation of laterality index from the random value (50%) for each of the dependent variables examined: index of angular position in the first 5 min (ang_index_5 or AI 5), index of parallel position in the first 5 min (par_index_5 or PI 5), total index (angular + parallel positions) in the first 5 min (index_5 or I 5), index of angular position in the second 5 min (ang_index_10 or AI 10), index of parallel position in the second 5 min (par_index_10 or PI 10), total index (angular + parallel positions) in the second 5 min (index_10 or I 10), global index (angular + parallel positions) in all 10 min of test (tot_index or Tot I). The results are summarized in the Table 1 and in Figs. 3 and 4. Results indicate the preference of the use of the left eye to look at the image in the mirror for fish reared in the natural LD condition (Fig. 3). By contrast, in all other Lighting Conditions there was no statistically significant value and therefore no expression of laterality in looking at the image in the mirror (Fig. 4). 4. Discussion
Fig. 2. Graph showing the percentage of the left eye use in different lighting conditions (group means with S.E.M.): LD = natural light/dark cycle, DD = continuous darkness, rLD = 14:10 red light-dark cycle, gLD = 14:10 green light-dark cycle, vLD = 14:10 violet light-dark cycle.
The lateralization (left eye use) that we measured on day 10 after hatching was evident only in those zebrafish that were exposed to natural light/dark conditions. Previously it had been shown [27] that a cycle
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Fig. 4. Presented here are the results of fry raised in the 14:10 LD cycles with monochromatic light of different wavelengths: (A) red, (B) green, and (C) violet light (rLD, gLD, vLD, respectively). The percentage of the left eye use is presented as group means with S.E.M., as in Fig. 3.
of 14 h of artificial white light and 10 h of darkness has, in the zebrafish larvae, the same effect of establishing lateralization of mirror-image viewing as we found here for the natural light/dark cycle (LD about 12:12). Keeping the developing embryos and the early post-hatching
zebrafish in darkness prevents the development of this asymmetry. Hence, lateralization of response by zebrafish manifested by seeing their image in a mirror requires exposure to white light in ovo and during early post-hatching development. After exposure to a cycle of 14 h of red, green or violet light and 10 h of darkness, no laterality of mirror-image viewing developed. It is, therefore, apparent that the neural events involved in development of this laterality require stimulation by a broad spectrum of light and that they do not develop after exposure to specific wavelengths alone. Certainly, red light alone, green light alone or violet light alone does not enable the development of this particular laterality. Since we controlled for the light intensity, we can be sure that a broad-spectrum of wavelengths is required, indicating that colour-coded neurons are involved. This result is quite similar to that found for the development of behavioural lateralization in chicks [16]. As mentioned in the Introduction, the lateralization in chicks develops after exposure of the embryo to white light, and to a lesser extent after exposure to green light, but not after exposure to red light (violet was not tested). It is interesting to note that the behavioural lateralization in both zebrafish and chicks differs from the demonstrated light-dependent asymmetry in the thalamofugal visual system of the chick. These neurons grow asymmetrically after stimulation by white, red or green light [16]. Presumably this mismatch between neuronal and behavioural response to light exposure depends on extra-visual neurons being used in control of the behaviour or in use of another visual pathway the development of which is colour-coded. In birds this could be the tectofugal visual system, which is known to respond to colour, whereas the thalamofugal visual system does not [33, 34]. However, Deng and Rogers [18] have shown that only the thalamofugal-recipient region of the chick forebrain is essential in performance of the pebble-grain task, and not the forebrain region that receives input from the tectofugal visual system. Hence, it seems that development colour-coded neurons outside the afferent visual input pathways to the forebrain are involved. This may be the case also in zebrafish. The zebrafish eye achieves its emmetropic form, typical of the adult, by as early as 72 h post-fertilization [35]. Prior to this, cone-visual ability has developed: cones develop by 40–50 h post-fertilization, bipolar cells by 60 h post-fertilization and both amacrine and horizontal cells by 50 h post-fertilization. These stages of development fall well within the period of exposure to different wavelengths of light that we used and so could potentially have been affected by the different treatments. By contrast, since retinal rod cells do not develop a functional role in vision until 15–40 dpf [36], much of their development occurs after the period of exposure used by us (our period of exposure was from fertilization to day 10 post-fertilization). Although very early stages of rod-cell differentiation could, perhaps, have been affected by exposure to different wavelengths of light, this seems less probable than an effect on cone cells. The tetrachromatic colour vision of the zebrafish depends on four types of cones [36]: viz., long-wavelength cones (L cones) with peak sensitivity in the red region of the spectrum (556–564 nm), M cones with a peak sensitivity in the green region (473–480 nm), S cones in the blue region (407–417 nm and these would be stimulated by the violet light that we used), and UV cones with their peak sensitivity in the ultraviolet region (360–361 nm). Our results indicate that more than one, and likely all four, of these types of cones must be stimulated during early development if lateralized visual responding to a mirror image is to develop. Stimulation of any one type of cone alone was not sufficient to generate the behavioural laterality. We used cyclic exposure to the different wavelengths of light, which was important since Robinson and Dowling [37] have shown that rearing zebrafish in constant light or dark leads to deficits in visual behaviour, even though these effects cause no detectable cellular deficits in retinal anatomy. In fact, timing-dependent activity of the retinal ganglion cells is necessary for refined development of retinotectal projections
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Table 1 Summary of the results. Statistical significant differences are in bold.
LD DD rLD gLD vLD
AI 5
PI 5
I5
AI 10
PI 10
I 10
Tot I
t11 = 3.65 p = 0.004 t10 = 0.93 p = 0.376 t9 = −0.30 p = 0.768 t10 = 1.54 p = 0.154 t8 = 0.89 p = 0.401
t11 = 1.84 p = 0.093 t10 = 1.33 p = 0.214 t9 = 1.96 p = 0.082 t10 = −0.54 p = 0.601 t8 = 0.58 p = 0.576
t11 = 3.42 p = 0.006 t10 = 1.28 p = 0.228 t9 = −0.63 p = 0.542 t10 = 0.59 p = 0.568 t8 = 0.98 p = 0.356
t11 = 2.53 p = 0.028 t10 = 0.24 p = 0.813 t9 = −0.02 p = 0.986 t10 = 1.71 p = 0.117 t8 = −0.55 p = 0.595
t11 = 3.14 p = 0.009 t10 = 0.13 p = 0.902 t9 = −0.40 p = 0.696 t10 = 0.66 p = 0.526 t8 = 0.83 p = 0.428
t11 = 3.23 p = 0.008 t10 = 0.36 p = 0.723 t9 = −0.24 p = 0.816 t10 = 1.72 p = 0.116 t8 = −0.16 p = 0.875
t11 = 4.31 p = 0.001 t10 = 0.74 p = 0.478 t9 = −0.81 p = 0.437 t10 = 1.38 p = 0.196 t8 = 1.51 p = 0.170
[38] and cyclic short-wavelength stimulation is necessary for development of UV sensitivity [39]. Such wavelength specificity is not apparent in the behavioural asymmetry that we investigated. The question arising now is whether the white-light dependent development of the behavioural lateralization is generated by asymmetry of eye stimulation, as known to be so in the case of the late embryonic effect of light on lateralization in chicks, or by light-dependent asymmetries in the timing of gene expression during development [40]. The latter is more likely to be the underlying mechanism since zebrafish eggs are transparent and, unlike chick embryos, the orientation of the embryo's body is unlikely to cause lateralized light stimulation. 5. Conclusions Asymmetry of the eyed use to view their image in a mirror is a characteristic of zebrafish raised in the natural photoperiod and in laboratory conditions in which they are exposed to a cycle of white light and darkness close to the natural cycle. Since no asymmetry develops if the zebrafish eggs and newly hatched fry are raised in the dark or are exposed to cycling monochromatic light (red, green or violet), we conclude that broad-spectrum stimulation is necessary to generate the neural asymmetry expressed as side differences in visual aspects of social behaviour. Acknowledgements This study was partly supported by an ERC Grant (ERC-2011ADG_20110406, Project No: 295517, PREMESOR) and by the University of Ferrara (FAR 2014). We wish to thank Lorella Fiorino for her help with the experiments and Andrea Margutti for the technical assistance. References [1] L.J. Rogers, Light experience and asymmetry of brain function in chickens, Nature 297 (1982) 223–225. [2] O. Güntürkün, The ontogeny of visual lateralization in pigeons, Ger. J. Psychol. 17 (1993) 276–287. [3] S. Budaev, R.J. Andrew, Shyness and behavioural asymmetries in larval zebrafish (Brachydanio rerio) incubated in the dark, Behavior 146 (2009) 1037–1052. [4] J.V. Zappia, L.J. Rogers, Light experience during development affects asymmetry of forebrain function in chickens, Brain Res. 313 (1983) 93–106. [5] L.J. Rogers, Evolution of hemispheric specialization: advantages and disadvantages, Brain Lang. 73 (2000) 236–253. [6] L.J. Rogers, Development and function of lateralization in the avian brain, Brain Res. Bull. 76 (2008) 235–244. [7] L.J. Rogers, Light input and the reversal of functional lateralization in the chicken brain, Behav. Brain Res. 38 (1990) 211–221. [8] M. Skiba, B. Diekamp, O. Güntürkün, Embryonic light stimulation induces different asymmetries in visuoperceptual and visuomotor pathways of pigeons, Behav. Brain Res. 134 (2002) 149–156. [9] R.J. Andrew, A.N. Johnston, A. Robins, L.J. Rogers, Light experience and the development of behavioural lateralisation in chicks. II. Choice of familiar versus unfamiliar model social partner, Behav. Brain Res. 5 (155) (2004) 67–76.
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