ZEBRAFISH Volume 11, Number 2, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/zeb.2013.0929
Circadian Clock Mediates Light/Dark Preference in Zebrafish (Danio Rerio) Ju Wang,1 Changhong Liu,1 Fei Ma,1 Wei Chen,1 Jian Liu,1 Bing Hu,2 and Lei Zheng1,3
Zebrafish (Danio rerio) has been a widely used vertebrate animal model in developmental biology and behavioral neuroscience, but knowledge about some of its basic behaviors, for example, light/dark preference, is still controversial. Appropriate preference for light or dark environments can be crucial for an animal’s survival, so we hypothesize that zebrafish may have its light/dark preference varied with the circadian clock. In this present work, we tested the hypothesis by recording the light/dark preference in a two-compartment tank continuously for over 60 h. We found that the light avoidance of fish generally increases with time from morning (8:00am) to midnight (2:00am), and then decreases with time from midnight (2:00am) to morning (8:00am), exhibiting a clear circadian-like trend in the light/dark preference. As melatonin can mediate circadian clock output and promote sleep in zebrafish, by adding extra melatonin at around 9:00am on the third day, the mean proportion of time fish spent in the dark area was increased significantly. Our results demonstrate that the circadian clock plays a significant role in regulating the light/dark preference in zebrafish, which provides valuable insights into understanding the metabolism mechanism underlying the neurobehavior, and facilitate further studies related to the neurobiology of normal and pathological behavior.
ebrafish (Danio rerio) play an integral role in biomedical research,1–4 enabling researchers to examine the physiological mechanisms and pathways relevant to behavioral neuroscience,5 as well as behavioral assays of drugs,6,7 human diseases,8 and genetic mutations.9 Very recently, a variety of behavioral assays have been developed to identify and characterize phenotypic changes, offering useful protocols for behavioral screens. For example, the light/dark preference test is one of the most commonly used anxiety models in animal preclinical studies10–15 and the approach has also been validated for screening both anxiolytic and anxiogenic drugs.14 Clearly, the appropriate preference for light or dark environments is vital to the survival of animals, as staying in a favorable environment can certainly help them to get food, to find conspecifics, or to avoid predators.16 So far, the light/ dark preference of zebrafish has been studied by many groups, however, no consensus is apparent with respect to this essential behavior in zebrafish.12–14,17 The behavioral features of zebrafish in a light/dark preference test was first reported by Serra et al.12 In addition, they found that in an 1 2 3
aquarium with two chambers, zebrafish exhibited a robust preference for the black compartment of the tank compared with the white one. Soon after, an apparently contradictory result was reported by Gerlai et al., which demonstrated that zebrafish significantly preferred the light compartment to a dark compartment in a similar preference task.17 Since then, this light/dark preference test has been replicated by different groups with their own experimental designs. Sackerman et al. claimed a clear black preference in the modified plus maze, with transparent walls and black/white floor.14 Champagne et al.13 then found that zebrafish showed a preference for the light chamber similar to Gerlai et al.’s. To explain the discrepancy, Blaser and Penalosa proposed that the differences in the apparatus, as well as stimuli, may affect zebrafish behavior in the light/dark preference test.18 Stephenson et al. considered that zebrafish preferred light or dark environments depending on the ambient light levels as well as olfactory stimulation.19 Apart from the above-mentioned explanations, we hypothesize that zebrafish may also have its light/dark preference varied with the circadian clock. As a diurnal species, zebrafish may prefer the light area during the daytime, while preferring the dark area during the
School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei, People’s Republic of China. School of Life Science, University of Science and Technology of China, Hefei, People’s Republic of China. School of Medical Engineering, Hefei University of Technology, Hefei, People’s Republic of China.
WANG ET AL.
nighttime, as their locomotor activities decrease and the arousal threshold increases.20 In the present work, we tested the hypothesis by recording the light/dark preference in a two-compartment tank continuously for over 60 h. Furthermore, melatonin, a hormone, is secreted by the pineal gland and retina in zebrafish,21 with high concentrations during the nighttime and low concentrations during the daytime, indicating that zebrafish have a robust circadian pattern of melatonin secretion.22,23 Interestingly, exogenous melatonin can also mediate circadian clock output and promote sleep in zebrafish.20 Thus, we chose melatonin to evaluate the effect of the circadian clock in zebrafish light/dark preference. Materials and Methods Materials
Subjects were adult wild-type (AB) zebrafish, of mixed sex group, raised in the laboratory from a line originally obtained from the University of Science and Technology of China (USTC) breeding facility. All the zebrafish used in this study were 1 year old. Individual fish was tested only once. The housing glass tank (34 · 19.5 · 23 cm, length · width · height) was filled with fresh system water identical to what was used for the maintenance of zebrafish. The number of fish housed together was about 10–15. Zebrafish were housed in the tank in a dedicated fish room. The water temperature was maintained at about 28C, with a 14-h light/10-h dark cycle (room fluorescent light, 08:00am–22:00pm). They were fed twice per day, at 09:00am and at 14:00pm, respectively, with freshly hatched brine shrimps. Apparatus
We tested previously untested zebrafish individually in a light or dark preference test. The apparatus consisted of a commercial fish tank (34 · 19.5 · 23 cm, length · width · height), an LED light panel, and a video camera with infrared feature (Fig. 1). To create a strong contrast in the light and dark environment, the preference tank was divided into two compartments, one chamber was covered with matte black paper on all sides and the top, and the other one was transparent. There was no physical barrier between the two compartments and the fish were allowed to swim freely in the entire fish tank. The LED light panel was placed above the experiment tank, ensuring that the light could cover the whole tank. An optical power meter (optical power meter SGN-II model; Tian Jin Shi Gang Dong Ke Ji Fa Zhan Corporation) was used to measure light intensity.24 Light levels inside the tanks ranged from 0.395 lW (in dark chambers) to 15.60 lW (in light chambers). To record the location and activity of zebrafish in the whole tank, a video camera with infrared feature (Wang Shi Wu You NVH-589MW; Wang Shi Wu You Corporation) was positioned of the testing tank, with *1m distance to both chambers. The surrounding environment was carefully controlled to ensure visual isolation of the fish from all direction. The video camera fed into a desktop computer with a user-specific fish tracking software, developed by Prof. John Y. Chiang, to track the swimming trajectory of zebrafish. To minimize the outside interference, the fish room was closed and kept quiet, and the experimenter was not visible to the fish during the recording.
FIG. 1. The apparatus was used in the experiment. The preference tank was divided into two compartments, one was illuminated by LED light and the other was covered with matte black paper on all sides and the top. The LED light was at the top of the experiment tank. A video camera was positioned in front of the tank. The behavior of zebrafish was recorded from 8:00am on the first day to 20:00pm on the third day. The horizontal white/black bars indicated subjective day versus subjective night, the horizontal striped bar indicated adding melatonin to fresh fish water between 9:00 and 20:00 during the third daytime, which ensures that the final concentration of melatonin in the tank was 0.1 mM.
For 0.1 mM stock solution, 0.2323 g melatonin (Aladdin) was directly dissolved in 10 mL ethanol. Then, a given volume of melatonin solution was added to water in the tank on day 3 to reach a final melatonin concentration of 10 nM, 0.001, and 0.1 mM, respectively. Procedure
To investigate the characteristic behavior of zebrafish, 54 untreated adult wild-type (AB) zebrafish were used for this light/dark preference test. The aquarium consisted of a dark chamber and a light chamber, and 10 L of fresh fish water was poured into the experimental tank, with a depth of 15 cm. Then, brine shrimps were added in the experimental tank at night before the trails, ensuring that the tank was filled with food uniformly and the food was sufficient for one fish for 3 days. All trials were performed and recorded for over 60 h, from 8:00am on day 1 till 20:00pm on day 3. Individual zebrafish was placed in the preference tank at 7:30am on day 1, and after a 30-min adaptation, the recording was started from 8:00am. After recording continuously for 2 days, we added a certain volume of melatonin to the fresh fish water at 9:00am on day 3, to reach a final melatonin concentration of 0, 10 nM, 0.001, and 0.1 mM, respectively. Previous study showed that ethanol has an anxiolytic effect on fish behavior,17 therefore, it is likely that ethanol may also affect the light/dark preference in zebrafish. To identify the effect of ethanol, we used the ethanol-only control group (n = 9) for comparison with the melatonin groups. In zebrafish, food anticipating and searching behavior may also influence the preference for light or dark. Zebrafish can
CIRCADIAN CLOCK MEDIATES LIGHT/DARK PREFERENCE
normally survive about 5–7 days without any food. To test this hypothesis, we also used the no-food control group (n = 9) to test the effect of food supply. The procedure for ethanol-only group and no-food group was otherwise the same as the normal experiment group. The groups with different treatments are shown in Supplementary Table S1 (Supplementary Data are available online at www.liebertpub.com/zeb). The temperature of the experimental room was controlled at 28C during all the experiments. The conditions between the home tank and observation tank are similar. Furthermore, other environmental factors, such as humidity, vibration, acoustic noise, illumination intensity and so on, were kept almost constant. Statistical analysis
All experimental data were analyzed by one-way analysis of variance (ANOVA), followed by post hoc comparisons between the experimental groups. Significance was set at p < 0.05. Data are presented as mean – SEM. Results
Figure 2 showed the effect of circadian variation on the preference for light or dark in zebrafish. On day 1, the fish displayed a preference for the light area of the aquarium between 8:00am and 18:00pm. The mean proportion of time fish spent in the light chamber of the tank was about 70.38% at 8:00am and about 52.78% at 18:00pm. As time went on, this preference was reversed to dark tendency when the time was 20:00pm, and the mean proportion of time in the dark was about 58.35%. After that, the mean proportion of time in the dark kept increasing with time till it achieved the maximum (74.06%) at 2:00am on day 2. Then, the mean proportion of time in the dark declined to the minimum value (43.53%) around 8:00am. From 8:00am day 2 to 8:00am day 3, fish showed exactly the same preference trend as the first 24 h. The mean proportion of time in the dark started from minimum (43.53%) at 8:00am on day 2, and kept increasing with time till it achieved the maximum (78.71%) at 2:00am on day 3. Subsequently, it decreased till around 8:00am. Thus, a clear circadian-like tendency of the proportion of staying in the dark area was observed in the 2 days.
A previous study showed that melatonin reduced the locomotor activities of fish and elevated their arousal threshold within 20 min after treatment.20 With addition of melatonin at 9:00am on day 3, the mean proportion of time in the dark with or without the effect of melatonin was investigated. About 0.1 mM melatonin could significantly increase the mean proportion of time in the dark to over 70% at 10:00am, much higher than that in the daytime on day 1 and 2 (below 60%). After 10:00am on day 3, zebrafish also spent more time in the dark area. These results indicated that melatonin could mediate the circadian clock of zebrafish, that is, a clear shift of daytime preference to nighttime preference on day 3. This provided strong evidence that the circadian clock played an important role in regulating the preference for light or dark in zebrafish. As the solvent of melatonin, ethanol is also an active compound and may change the behavior of zebrafish. We tested this by adding the same amount of ethanol, but without melatonin on day 3. As seen from Figure 3, there was a significant difference between the melatonin group and nomelatonin group at 10:00am on day 3 (F1,16 = 5.75, p < 0.05), indicating that, unlike melatonin, ethanol itself could not incur any notable change in the light/dark preference behavior of zebrafish. In other words, ethanol used in this study did not change the circadian clock in light/dark preference in zebrafish. To check the effect of feeding, as seen from Figure 4, zebrafish from the no-food group were still able to display similar circadian cycling of light/dark preference compared with the food group, for example, the light avoidance of fish generally increased with time from morning (8:00am, 37.97%) to midnight (2:00am, 87.97%), and then decreased with time from midnight (2:00am, 87.97%) to morning (8:00am, 50.92%) between day 1 and 3, even though the mean proportion of time in the dark for the no-food group was generally a bit higher compared with the food group. There was no significant difference ( p > 0.05) between the two groups during the whole experiment except several time points (Fig. 4), this was probably attributed by the individual difference. Overall, the two groups have similar circadianlike trend in the light/dark preference, suggesting that the feeding could hardly affect the circadian clock in light/dark preference in zebrafish.
FIG. 2. Plot of the proportion of time spent in the dark at each time of day in zebrafish. Horizontal white/black bars indicate subjective light versus subjective night according to the 14:10 light–dark cycle of zebrafish. The melatonin was added between 9:00 and 20:00 on the third day. The behavior of zebrafish was recorded by the video camera. Each data point represents mean – SEM. n = 9 for group.
WANG ET AL.
FIG. 3. Plot of the proportion of time spent in the dark area at each time of day in zebrafish when adding no melatonin (closed squares) compared with adding melatonin (closed diamonds) on the third morning. Each data point represents mean – SEM. n = 9 for melatonin group, n = 9 for no melatonin group. The asterisk denotes significant difference between no melatonin group and melatonin group at 10:00am on day 3 ( p < 0.05). To gain a further understanding of whether melatonin influences the behavior of zebrafish is dose dependent, multiple concentrations of melatonin (0, 10 nM, 0.001, and 0.1 mM) were selected in this study (Supplementary Table S2 and Fig. 5). The four individual groups had similar light/dark preference ( p > 0.05) during the first 2 days, and they all exhibited a robust circadian trend in the light/dark preference, but at 10:00am on day 3 after different concentrations of melatonin treatment, four individual groups displayed obvious differences. For example, the zebrafish in the higher concentration of melatonin (0.1 mM) spent more time in the dark area compared with the nothing group (F1,16 = 5.56, p < 0.05) and the 10 nM melatonin group (F1,16 = 8.95, p < 0.01). However, the lower concentration of melatonin groups (including 10 nM melatonin group and 0.001 mM melatonin group) and nothing groups did not show any difference ( p > 0.05) between each other at 10:00am on day3. The comparison between the four melatonin groups (0, 10 nM, 0.001, and 0.1 mM) is shown in Supplementary Table S2. Discussion
As a typical species, zebrafish can perceive the alteration from day to night, thus, the circadian variation has to be considered for the relevant studies. The light/dark preference of zebrafish has been studied previously by many groups, but little attention has been paid to the intraday variation of the
experiments.12–14,17 For examples, Serra et al.12 and Gerlai et al.17 did not mention the specific time for their tests. Stephenson et al. performed their experiments only between 9:30am and 4:30pm over 3 days.19 As far as we know, zebrafish is a typical diurnal animal, which is well accommodated to both light and dark environments, enabling it to react immediately to the illumination condition. Even zebrafish larvae can perceive the variation in the external environment, altering the distribution of melanin pigment in their skin according to ambient light levels and to match backgrounds.25 Moreover, zebrafish adults displayed higher activities during the daytime, as they need to detect predators, to get food or to find conspecifics.16 In the nighttime, fish displayed brief periods of inactivity, often associated with a drooping caudal fin, suggesting a sleep-like state,26 Consequently, zebrafish are generally more active with a preference of light area during the daytime, while during the nighttime, they prefer dark backgrounds to minimize their visibility and their locomotor activities.27 Our results imply that the circadian clock may be a fundamental factor to decode the mystery of many complicated animal behaviors. In the present study, during the first few hours of the trial, fish appeared to prefer the light area of the tank, probably because they need to be prepared to behave in the daytime, for example, looking for the food and conspecifics. More time in the light area might help them keep alert and stay active. As time went on, the light avoidance
FIG. 4. Plot of the proportion of time spent in the dark area at each time of day in zebrafish when adding nothing (closed squares) compared with adding food (closed diamonds) in the whole experiment. Each data point represents mean – SEM. n = 9 for food group, n = 9 for no food group.
CIRCADIAN CLOCK MEDIATES LIGHT/DARK PREFERENCE
FIG. 5. Plot of the proportion of time spent in the dark area at each time of day in zebrafish in different concentrations of melatonin [nothing (closed diamonds), 10 nM (closed squares), 0.001 mM (closed triangle), and 0.1 mM (closed circle)]. Each data point represents mean – SEM. n = 9 for four groups. The asterisk denotes significant difference between zero group and 0.1 mM melatonin group at 10:00am on day 3 ( p < 0.05). The two asterisks denote an extremely significant difference between 10 nM melatonin group and 0.1 mM melatonin group during adding melatonin on day 3 ( p < 0.01).
behavior of fish started to increase, and the mean proportion of time in the dark reached the maximum around 2:00am, possibly because zebrafish need to gradually switch themselves from daytime mode to nighttime mode. More time in the dark area might help them create melatonin to promote sleep. The circadian clock system is a phylogenetically highly conserved mechanism, which is temporal synchronization of the biological processes with the regular 24-h changes of our planet environment.28 Zebrafish are diurnal vertebrates with clear daytime activity and nighttime rest in relation to the robust circadian pattern of melatonin secretion.22,23 For example, in zebrafish, their locomotor activities were reduced and their arousal threshold was elevated during the constant darkness,20 while the opposite phenomena were observed when they were in the daytime. It has been reported that the pineal organ and retina contain circadian oscillators that regulate rhythmic melatonin production. For instance, pineal melatonin release peaked during the subjective night and returned to low levels during the subjective day, because pineal melatonin production was suppressed acutely during light exposure.21 To confirm the effect of circadian clock on zebrafish light/dark preference, we chose melatonin as a factor of circadian variation, our results showed that extra melatonin uptake in the daytime quickly shifted zebrafish from the daytime mode to the nighttime mode, for example, zebrafish spent the mean proportion of time which reached 70% in the dark area even though in the daytime. Therefore, we concluded that melatonin could regulate the circadian clock of zebrafish, and proved that the circadian clock mediated the preference for a light or a dark environment in zebrafish. The effects of alcohol could be observed in multiple behavioral paradigms. Alcohol mixed in the water of fish can be easily absorbed by the blood vessels of the gill and the skin of the fish, so that blood alcohol levels reach equilibrium with the external alcohol concentration quickly.29 In the test of light/dark preference, higher doses of alcohol decreased habituation of zebrafish to a dark compartment in a light/dark choice paradigm.17 Therefore, ethanol, as the solvent of melatonin, is an active compound and may change the behavior of zebrafish. Whereas from our result, the final con-
centration of ethanol in the tank was very low, it could not change the behavior of zebrafish. Additionally, it is difficult to distinguish between the behavior and the effect of food in the light/dark preference in zebrafish. People reported that rats increased their locomotor activity 2–4 h before the onset of food availability when food was available only for a limited time each day.30 Moreover, the food also influenced the behavior of zebrafish. For example, in trout, the time of feeding has been found to affect the daily rhythms of plasma hormones and metabolites.31,32 Therefore, food searching behavior may also influence the circadian clock on zebrafish light/dark preference. However, the no-food group, in the present result, displayed the same circadian-like trend in the light/dark preference compared with the food group, so feeding could hardly affect the circadian clock in light/dark preference in zebrafish. It is reported that the half-life of melatonin is very short, for example, the mean melatonin half-life was 57 – 34 min in plasma samples in humans.33 As for rats, the total amount of melatonin was decreased in all tissues after 30 min.34 So, we chose the effect of melatonin at 10:00am on day 3 mainly for evaluating the light/dark preference. The different concentrations of melatonin made zebrafish display various behaviors. For instance, at 10:00am on day 3 after melatonin treatment, zebrafish in 0.1 mM melatonin spent more time in the dark area compared with nothing and 10 nM melatonin groups, but the lower concentration of melatonin groups (including 10 nM and 0.001 mM melatonin groups) and the nothing group had no obvious changes in their behavior. These results suggest that the behavior of zebrafish influenced by melatonin is dose dependent. At 12:00am and 20:00pm on day 3, the light/dark preference among the three groups (nothing group, 0.001, and 0.1 mM melatonin groups) did not show any difference, this was probably attributed that melatonin had been metabolized and the concentration of melatonin was very low in blood of zebrafish, thus it could hardly affect the zebrafish behavior. In summary, we demonstrated that circadian clock plays a significant role in regulating the light/dark preference in zebrafish. These results, as well as the previous studies by Serra et al.,12 Gerlai et al.,17 and Stephenson et al.19 indicated that circadian variation needed to be considered to design the
preference test and compare the results between different groups. It is also worth emphasizing that the circadian clock might also affect many other cognitive and neural behaviors, which might be useful for validating the model system for drug screening and human disease studies.14 Our findings will provide new insights in understanding the metabolism mechanism underlying the neurobehavior, and facilitate further studies related to the neurobiology of normal and pathological behavior. Acknowledgments
We thank Prof. John Y. Chiang for providing us the userspecific fish tracking software. This study is supported by the specialized Research Fund for the Doctoral Program of Higher Education (20120111110024), the Fundamental Research Funds for the Central Universities (2012HGCX0003), (2012HGZY0021), the National Key Technologies R&D Programme (2012BAD07B01), and the Funds for Huangshan Professorship of Hefei University of Technology. Disclosure Statement
No competing financial interests exist. References
1. Moretz JA, Martins EP, Robison BD. Behavioural syndromes and the evolution of correlated behaviour in zebrafish. Behav Ecol 2007;18:556–562. 2. Spence R, Gerlach G, Lawrence C, Smith C. The behaviour and ecology of the zebrafish, Danio rerio. Biol Rev 2008; 83:13–34. 3. Speedie N, Gerlai R. Alarm substance induced behavioural responses in zebrafish (Danio rerio). Behav Brain Res 2008;188:168–177. 4. Gumm JM, Snekser JL, Iovine MK. Fin-mutant female zebrafish (Danio rerio) exhibit differences in association preferences for male fin length. Behav Process 2009;80: 35–38. 5. Sison M, Gerlai R. Associative learning in zebrafish (Danio rerio) in the plus maze. Behav Brain Res 2010;207:99–104. 6. Darland T, Dowling JE. Behavioural screening for cocaine sensitivity in mutagenized zebrafish. Proc Natl Acad Sci U S A 2001;98:11691–11696. 7. Gerlai R, Lee V, Blaser R. Effects of acute and chronic ethanol exposure on the behaviour of adult zebrafish (Danio rerio). Pharmacol Biochem Behav 2006;85:752–761. 8. Lieschke GJ, Currie PD. Animal models of human disease: zebrafish swim into view. Nat Rev Genet 2007;8:353–367. 9. Itzkowitz M, Iovine MK. Single gene mutations causing exaggerated fins also cause non-genetic changes in the display behaviour of male zebrafish. Behaviour 2007;144: 787–795. 10. Maximino C, Marques deBrito T, Dias CAGdM, Gouveia A, Morato S. Scototaxis as anxiety-like behaviour in fish. Nat Protoc 2010;5:209–216. 11. Maximino C, Marques deBrito T, Colmanetti R, Pontes AAA, Meira deCastro H, Tavares deLacerda RI, et al. Parametric analyses of anxiety in zebrafish scototaxis. Behav Brain Res 2010;210:1–7. 12. Serra EL, Medalha CC, Mattioli R. Natural preference of zebrafish (Danio rerio) for a dark environment. Braz J Med Biol Res 1999;32:1551–1553.
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13. Champagne DL, Hoefnagels CCM, de Kloet RE, Richardson MK. Translating rodent behavioural repertoire to zebrafish (Danio rerio): relevance for stress research. Behav Brain Res 2010;214:332–342. 14. Sackerman J, Donegan JJ, Cunningham CS, Nguyen NN, Lawless K, Long A, et al. Zebrafish behaviour in novel environments: effects of acute exposure to anxiolytic compounds and choice of Danio rerio line. Int J Comp Psychol 2010;23:43–61. 15. Costall B, Jones BJ, Kelly ME, Naylor RJ, Tomkins DM. Exploration of mice in a black and white test box: validation as a model of anxiety. Pharmacol Biochem Behav 1989;32:777–785. 16. Frey H. Das Aquarium Von a Bis Z. Radebeul: Neuman Verlag, 1966. 17. Gerlai R, Lahav M, Guo S, Rosenthal A. Drinks like a fish: zebra fish (Danio rerio) as a behaviour genetic model to study alcohol effects. Pharmacol Biochem Behav 2000;67: 773–782. 18. Blaser RE, Penalosa YM. Stimuli affecting zebrafish (Danio rerio) behavior in the light/dark preference test. Physiol Behav 2011;104:831–837. 19. Stephenson JF, Whitlock KE, Partridge JC. Zebrafish preference for light or dark is dependent on ambient light levels and olfactory stimulation. Zebrafish 2011;8: 17–22. 20. Zhdanova IV, Wang SY, Leclair OU, Danilova NP. Melatonin promotes sleep-like behaviour in zebrafish. Brain Res 2001;903:263–268. 21. Cahill GM. Circadian regulation of melatonin production in cultured zebrafish pineal and retina. Brain Res 1996;708: 177–181. 22. Kazimi N, Cahill GM. Development of a circadian melatonin rhythm in embryonic zebrafish. Dev Brain Res 1999;117:47–52. 23. Cahill GM, Hurd MW, Batchelor MM. Circadian rhythmicity in the locomotor activity of larval zebrafish. Neuroreport 1998;9:3445–3449. 24. Pan WJ, Xiong C, Wu QP, Liu JX, Liao HM, Chen W, et al. Effect of BPA on the germination, root development, seedling growth and leaf differentiation under different light conditions in Arabidopsis thaliana. Chemosphere 2013;93:2585–2592. 25. Fleisch VC, Neuhauss SCF. Visual behaviour in zebrafish. Zebrafish 2006;3:191–201. 26. Yokogawa T, Marin W, Faraco J, Pezeron G, Appelbaum L, Zhang J, et al. Characterization of sleep in zebrafish and insomnia in hypocretin receptor mutants. PLoS Biol 2007;5:e277. 27. Gerlai R. Zebrafish antipredatory responses: a future for translational research? Behav Brain Res 2010;207:223–231. 28. Zhdanova IV, Yu L, Lopez-patino M, Shang E, Kishi S, Guelin E. Aging of the circadian system in zebrafish and the effects of melatonin on sleep and cognitive performance. Brain Res Bull 2008;75:433–441. 29. Ryback R, Percarpio B, Vitale J. Equilibration and metabolism of ethanol in the goldfish. Nature 1969;222:1068– 1070. 30. Richter CP. A behavioristic study of the activity of the rat. Comp Psychol Monogr 1922;1:1–5. 31. Reddy PK, Leatherland JF. Does the time of feeding affect the diurnal rhythms of plasma hormone and glucose concentration and hepatic glycogen content of rainbow trout? Fish Physiol Biochem 1994;13:133–140.
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32. Reddy PK, Leatherland JF. Influence of the combination of time of feeding and ration level on the diurnal hormone rhythms in rainbow trout. Fish Physiol Biochem 1995;14:25–36. 33. Claustrat B, Brun J, Garry P, Roussel B, Sassolas G. A once-repeated study of nocturnal plasma melatonin patterns and sleep recordings in six normal young men. J Pineal Res 1986;3:301–310. 34. Kopin IJ, Pare CMB, Axelrod J, Weissbach H. The fate of melatonin in animals. J Biol Chem 1961;236:3072–3075.
Address correspondence to: Lei Zheng, PhD School of Biotechnology and Food Engineering Hefei University of Technology No. 193 Tunxi Road Hefei 230009 People’s Republic of China E-mail: [email protected]
; [email protected]