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Neurotoxicology and Teratology

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ACCEPTED MANUSCRIPT Effects of Diphenylhydantoin on Locomotion and Thigmotaxis of Larval Zebrafish a

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Xiuyun Liu , Jia Lin , Yinglan Zhang , Xiaolan Peng , Ning Guo , Qiang Li a

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Translational Medical Center for Development and Disease, Shanghai Key Laboratory of Birth Defect, Institute of Pediatrics, Children’s Hospital of Fudan University, 399 Wanyuan Road, Shanghai 201102, China b Center for Chinese Medical Therapy and Systems Biology, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai 201203, China

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Please direct all correspondences to: Qiang Li, Translational Medical Center for Development and Disease, Shanghai Key Laboratory of Birth Defect, Institute of Pediatrics, Children’s Hospital of Fudan University, 399 Wanyuan Road, Shanghai 201102, China; Tel.: +86-21-64931011, Fax: +86-21-64931011, E-mail address: [email protected] Ning Guo, Center for Chinese Medical Therapy and Systems Biology, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai 201203, China; Tel.: +86-21-51322748, Fax: +86-21-51322642, Email: [email protected]

Abstract

Antiepileptic effects of Diphenylhydantoin (DPH) have been documented in animal studies and

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clinical research, while little is known about the effects of the drug on basic behaviors and anxiety-related behaviors. In order to understand neuroactivities of DPH deeply and administrate DPH in clinic rationally, it is necessary to study neurobehavioral effects of the drug. In the present study, the effects of DPH on the locomotor activity and thigmotaxis of zebrafish larvae at 5 days post fertilization

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(dpf) were explored under different illumination conditions. The influence of DPH on zebrafish larval responses to visual stimuli (sudden illumination transition from light to dark) was also investigated.

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Under light or dark condition, exposure to high concentrations of DPH resulted in decreased locomotor activity and thigmotaxis, whereas DPH treatment at low doses enhanced the locomotor activity.

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Additionally, sudden illumination transition induced robust increase in the locomotor activity and this phenomenon was not modified by DPH treatment. Our results suggest that DPH has potential stimulatory and inhibitory effects on the locomotor activity and possesses anxiolytic properties. In addition, responses of 5-dpf zebrafish larvae to visual stimuli were not modified by DPH treatment.

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Keywords

Diphenylhydantoin, Zebrafish larva, Behavior, Locomotor activity, Thigmotaxis 1.

Introduction Zebrafish (Danio rerio), as an emerging non-mammalian vertebrate model organism, is ideally

suited for large-scale analyses of drug-induced behaviors. Hundreds of fertilized eggs can be readily harvested daily and can be immediately and directly treated with external compounds during different developmental stages and their small size allows for easier handling and husbandry (Colwill and Creton 2011; Richendrfer, Pelkowski et al. 2012). Zebrafish develop rapidly into free-swimming larvae. A fertilized egg develops into a larva with eyes, a beating heart, and tail movements after 24 hours (Kimmel, Ballard et al. 1995). By 4–5-dpf, zebrafish larvae demonstrate a broad range of behaviors, such as hunting, avoidance, startle response, scototaxis and thigmotaxis (Fetcho and Liu 1998; Colwill and Creton 2011; Schnorr, Steenbergen et al. 2012). Thus, many behavioral tests can be carried out in larval zebrafish as early as the first week post fertilization. Thigmotaxis, as an index of anxiety, is the unconditioned preference of an animal for the boundaries of an environment. This behavior is evolutionarily conserved and performed by a variety of species, including humans (Kallai, Makany et al. 2005; Kallai, Makany et al. 2007), rodents (Treit and

ACCEPTED MANUSCRIPT Fundytus 1988; Prut and Belzung 2003; Sousa, Almeida et al. 2006; Belzung and Philippot 2007) and fish (Peitsaro, Kaslin et al. 2003; Lopez-Patino, Yu et al. 2008; Sharma, Coombs et al. 2009; Champagne, Hoefnagels et al. 2010; Colwill and Creton 2011). Zebrafish larvae display clear thigmotaxis behavior as well. Many factors are able to influence the thigmotaxis of zebrafish larvae,

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such as feeding and neuroactive drugs. The fed 7-dpf larvae displayed a decreased thigmotaxis, compared with the unfed 7-dpf larvae (Clift, Richendrfer et al. 2014). The thigmotaxis of 5-dpf larvae

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was significantly attenuated by the anxiolytic agent diazepam and significantly enhanced by the anxiogenic drug caffeine (Schnorr, Steenbergen et al. 2012). In addition, Zebrafish larvae at 5-dpf

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exhibited reduced thigmotaxis when treated with yohimbine at 25 mg/L, while higher concentrations of yohimbine have no effect on the thigmotaxis in 5-dpf larvae (Li, Lin et al. 2014). DPH, a sodium channel blocker, is one of the most popular antiepileptic drugs (Munro, Erichsen et al. 2007). It is supposed to act by modification of glutamatergic transmission (Bremner, Mletzko et al.

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2005). Although DPH has good pharmacological efficacy, its clinical use is often limited because of side effects such as teratogenicity and neurotoxicity. Teratogenicity effects of DPH have been documented in studies with rodents and humans (Cohn, Axelrod et al. 1978; Yang, Chi et al. 1978; Kim,

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Lee et al. 2012), while more attention should be paid on functional deficiencies study, such as neurobehavioral function, as behavioral analysis is perhaps the best way to investigate the function of the brain (Gerlai 2003; Tierney 2011). The neuroactivities of DPH have been studied in rodents. Pregnant Sprague-Dawley (SD) rats orally administered DPH (200 mg/kg) on days 7-18 of gestation

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showed increased early locomotor activity in offspring (Minck, Acuff-Smith et al. 1991). The

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intraperitoneal injection of DPH at 5–10 mg/kg blocked the increased open-field locomotion that was induced by methylphenidate (Tonelli, Pereira et al. 2013). The daily pretreatment of the DPH (120 mg/kg, i.p.) abolished the locomotor hyperactivity in the open-field test in rats of seizures induced by

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repeated electroconvulsive shock (ECS) (Hidaka, Suemaru et al. 2008). Adult SD rats that were exposed to therapeutic levels of DPH throughout prenatal development and the postnatal pre-weaning period showed increased rates of acquisition and performance in both the appetitive and avoidance learning paradigms and a substantial impairment in avoidance learning following the transfer from

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appetitive to aversive conditioning (Mowery, McDowell et al. 2008). Male Wistar rats that were exposed to DPH (75 mg/kg, i.p.) for 21 days displayed significant deficits in learning/memory as indicated by a significant increase in the retention transfer latency in an elevated plus maze test and a significant decrease in the retention latency in the passive avoidance paradigm (Reeta, Mehla et al. 2009). In addition to the work in rodents, there are relatively few reports on the locomotion effects of DPH in zebrafish and most were relative to seizure. The seizure-related behavioral alterations in adult zebrafish that were induced by pentylenetetrazole (PTZ) treatment were suppressed by pretreatment with DPH at 450 µM for 1 hour (Siebel, Piato et al. 2013). The seizure-like swimming pattern in zebrafish larvae that was induced by 2 hours exposure to ginkgotoxin was significantly reversed by simultaneously adding 1 mM of DPH (Lee, Sung et al. 2012). AEDs have been successfully used in the treatment of mood disturbances including anxiety disorders, such as tiagabine (Baetz and Bowen 1998) , gabapentin (Pollack, Matthews et al. 1998) and pregabalin (Stahl 2003). DPH treatment (30 mg/kg, i.p.) for 15 days significantly improved the depressive like behavior along with its anticonvulsant effect in Male Swiss Albino mice that were successfully kindled by pentylenetetrazole (Choudhary, Mishra et al. 2013). In Male ddY mice at 7-8 weeks of age, DPH treatment (10 mg/kg, i.p.) enhanced effects of (+)-SKF- 10,047 and

ACCEPTED MANUSCRIPT dextromethorphan on the stress response, while the effects could be blocked by the dopamine D1 and D2 receptor antagonists (Kamei, Kameyama et al. 1996). DPH has been demonstrated to exert prophylactic effect in bipolar disorder patients, one of the most common mood disorders (Mishory, Winokur et al. 2003).

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It has been implied from the experimental literature that DPH exposure could influence behaviors, such as learning- and seizure-related behaviors, while little is known about its effects on basic

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behaviors and anxiety-related behaviors. Study of neurobehavioral effects of the drug is necessary for better understanding of neuroactivities of DPH and beneficial for the rational administration of DPH in

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clinic. In this current study, we explored the effects of DPH on the locomotor activity and thigmotaxis of zebrafish larvae at 5-dpf under light or dark conditions, because behaviors of zebrafish can be influenced by environmental factors (Irons, MacPhail et al. 2010; de Esch, van der Linde et al. 2012). The influence of DPH on zebrafish larval responses to visual stimuli was also investigated. It was larvae, which would be concentration- dependent. Materials and Methods

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hypothesized that DPH could modify the locomotor activity and anxiety-related behavior in zebrafish

2.1 Animals and housing

AB wild type zebrafish were maintained at 28.5 oC and kept on a 14:10-hour light:dark cycle (lights on at 08:00 AM). The eggs were obtained by random mating between sexually mature individuals and

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were raised in groups of 50 in an incubator at 28.5 oC. The eggs and larvae were kept under the same

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lighting schedule as that of adult zebrafish. Larvae were allowed to develop under this condition until behavioral testing at 5-dpf and the larvae that were used in the experiments were not fed. All of the

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animal experimental procedures complied with local and international regulations. All of the protocols were approved by the institutional animal care committee, Children’s Hospital of Fudan University. 2.2 Drugs

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5, 5-Diphenylhydantoin sodium salt (D4505-25G, Sigma-Aldrich) was dissolved in a 30 mM stock solution with sterilized water and stored at -80 oC. DPH working solution was freshly diluted from stock solution to appropriate concentrations with zebrafish system water before the experiments. 2.3 Drug treatment and behavior tests Behavior tests were carried out with the zebrafish larvae at 5-dpf in 24-well plates. All the experiments were performed 2 hours after the beginning of the light cycle and 2 hours before the beginning of the dark cycle. The experiments were arranged in a way that all concentration groups were equally presented in each 24-well plate to avoid any inter-treatment variations due to differences in experiment timing during the day. The zebrafish larvae were carefully transferred to a 24-well plate with one single larva in each well. Excessive fluid was removed, and 500 µL of fresh system water was loaded into each well immediately. Subsequently, 500 µL of DPH working solution was quickly added into the wells; therefore, each well contained 1 mL liquid. The final tested DPH concentrations were 0 (control), 1, 4, 20, 100 and 500 µM. The plate was then placed into Zebrabox (ViewPoint Life Sciences) equipped with a recorder to record the video of zebrafish larvae activities. The current protocol and experimental procedure are shown in Fig. 1A.

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2.3.1 Locomotor activity The quantification of zebrafish larvae locomotor activities was achieved using the tracking mode of Zebralab software with recorded videos. The videos of zebrafish larvae were taken at the rate of 25 fps,

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and were pooled into 1 min time bins. Only the total distance travelled was obtained for analysis. The distance moved by the larvae in the whole well was acquired for the analysis of general locomotor

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activities.

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2.3.2 Thigmotaxis

A round-shaped center arena that occupied half of the area of a single well was defined in each well (Fig. 1B). Thigmotaxis was presented as the percentage (%) of the total distance moved (TDM) in the outer zone of the test apparatus as previously described by Schnorr et al (Schnorr, Steenbergen et al.

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2012). The percentage of TDM in the outer zone was obtained by multiplying this ratio by a factor of 100 as depicted in the formula below. This calculation was performed to correct for individual differences in the locomotor activity as recommended by Bouwknecht and Paylor (Bouwknecht and

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Paylor 2008).

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2.4 Data presentation and statistics analysis

Data are presented as the mean ± SEM. Statistical analyses and graphs were performed using GraphPad Prism software (version 5.0).

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One-way ANOVA followed by Dunnett’s multiple comparison post hoc tests were performed to compare the DPH-treated groups with the controls to assess the effects of DPH on the locomotor activity and thigmotaxis, and a probability level of 5% was used as the minimal criterion of significance.

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Student’s t-tests (two-tailed) were performed to analyze the behavioral changes in response to light/dark challenges within each concentration group (light vs. dark). The minimal criterion of significance was set at 5%. Correlation analysis was performed between the locomotor activity and thigmotaxis in the same DPH-treated groups and corresponding control groups. 3.

Results

3.1 Effects of DPH on the locomotor activity of zebrafish larvae under light condition. Distance moved in each 1-min time bin by larvae during 35-min measurements period (10-min to 45-min) within the entire zone (one entire well of the 24-well plate) were plotted against the progression of the experiment and different dose groups were presented with the control group one at a time (Fig. 2). To better characterize the effect of DPH treatment on the locomotor activity of zebrafish larvae, average distance moved per minute by larvae under 30-min light condition were analyzed. DPH treatment at concentrations up to 20 µM produced a gradual increase in the locomotor activity, while DPH treatment at higher concentrations resulted in reduced locomotor activity, although only the 500 µM DPH treated group was significantly different from the non-treated control group (Fig. 3). The results suggest that the effect of DPH on the locomotor activity was concentration- dependent and DPH

ACCEPTED MANUSCRIPT exerted an inhibitory effect at high doses. 3.2 Effects of DPH on the thigmotaxis of zebrafish larvae under light condition. In addition to the effect of DPH on the locomotor activity, the influence of DPH on the thigmotaxis

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was studied as well. Thigmotaxis was presented as the percentage (%) of the total distance moved in the outer zone of the test apparatus (%TMD in the outer zone) (Fig. 4). As shown in Fig. 1B, each well

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was equally divided into inner and outer zones. Therefore, if no thigmotaxis behavior was observed, the total distance moved in the outer zone would account for 50% of the activities in the whole well. It was

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obvious that in the non-treated groups, zebrafish larvae demonstrated clear thigmotaxis, as the %TMD in the outer zone was much greater than 50%.

For the DPH treated groups, no differences in thigmotaxis were observed in groups treated with DPH at or lower than 20 µM compared with the control group. Starting from 100 µM, a reduction in

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thigmotaxis was observed. Although the 100 µM and the 500 µM DPH treated groups demonstrated similar levels of thigmotaxis, only the 100 µM treated group was significantly lower than the control group (Fig. 4). The results suggested that DPH possessed anxiolytic properties in zebrafish larvae at

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high concentrations.

3.3 Effects of DPH on the locomotor activity of zebrafish larvae under dark condition. Under dark condition, compared with the control group, larvae receiving DPH treatment at 20 µM

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demonstrated a significantly increased locomotor activity, whereas DPH treatment at 500 µM caused a

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significant decrease in the locomotor activity (Fig. 5). This was similar to what was observed under light condition, although the increase in locomotor activity caused by 20 µM DPH treatment under

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light condition was not statistically significant (Fig. 3). 3.4 Effects of DPH on the thigmotaxis of zebrafish larvae under dark condition. Similar to the phenomenon that was observed during the light phase, during the dark phase, zebrafish larvae demonstrated clear thigmotaxis (Fig. 6). In addition, DPH treatment at 500 µM

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significantly attenuated the thigmotaxis in the dark, while all other lower concentrations that were tested in this study did not exert significantly influences on thigmotaxis. 3.5 Effects of DPH on zebrafish larval responses to abrupt illumination transition. The locomotor responses of zebrafish larvae to a sudden illumination change from light to dark under the influence of DPH were also examined. Different dose groups were plotted with the control group one at a time (Fig. 2). As shown in previous studies, zebrafish larvae in control groups exhibited an immediate and robust increase in the locomotor activity when illumination was abruptly switched from light to dark. Although the robust increase was not affected by DPH treatment, different response patterns to light/dark transition were observed at different concentrations (Fig. 2). To better characterize the effect of DPH treatment on the locomotor activity of zebrafish larvae in response to light/dark challenge, the distance moved in the minute before and the minute after sudden illumination transition representing the moment before and the moment after sudden light change respectively was analyzed (Fig. 7). In the non-treated groups, when given an illumination transition from light to dark, the locomotor activity of zebrafish larvae increased significantly, as the distance moved in the minute after sudden illumination transition significantly increased compared to that before sudden illumination transition. Our results suggest that illumination challenge from light to dark

ACCEPTED MANUSCRIPT introduced a dramatic increase in the locomotor activity of zebrafish larvae. The phenomenon of illumination transition introducing a robust increase in the locomotor activity was observed in all of the DPH-treated groups, because the distance moved after light change was significantly higher than that before light change in all of the DPH-treated groups (Fig. 7). These results indicated that DPH did not

Discussion

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eliminate zebrafish larval responses to illumination challenge from light to dark.

In this study, the behavioral responses of zebrafish larvae to DPH treatment were evaluated. To

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better illustrate the effects of DPH on zebrafish behaviors, different doses of DPH were analyzed. Different lighting conditions were considered as well. In the non-treated groups, zebrafish larvae displayed a relatively low locomotor activity under light condition, while a relatively high activity was observed under dark condition. The same phenomenon was previously reported, as zebrafish larvae at

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5-, 6- and 7-dpf showed a relatively low activity in light while in darkness a relatively high activity was observed (Padilla, Hunter et al. 2011; de Esch, van der Linde et al. 2012) . Given that DPH could influence behaviors in rodent (Tonelli, Pereira et al. 2013), DPH modified

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the locomotor activity of zebrafish larvae. This effect was not influenced by light conditions, but it was affected by the concentrations of DPH. Under light or dark conditions, DPH exerted a stimulatory effect at low doses but an inhibitory effect at high doses in zebrafish larvae. The results suggested that different signal pathways could engage in the effect, which needs more detailed work. The

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dopaminergic receptor is important in the regulation of the locomotor activity, because dopaminergic

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receptor agonists increase the locomotor activity, while antagonists decrease the locomotor activity in larval zebrafish (Padilla, Hunter et al. 2011). These results indicate that alteration in dopaminergic signaling could play a role in the behavioral changes in zebrafish. Our results again confirmed the fact

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that zebrafish are suitable for behavioral testing as early as 5-dpf. DPH is a common antiepileptic drug with varieties of neuroactivities, such as antimanic effect (Tonelli, Pereira et al. 2013), antidepressant-like effect (Choudhary, Mishra et al. 2013) and so on. In the current work, DPH had an anxiolytic effect in zebrafish larvae and the effect was

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concentration-dependent. Under light or dark conditions, DPH exerted an anxiolytic effect at high concentrations while showing neither anxiolytic nor anxiogenic properties at low concentrations. One alternative explanation would be that high concentrations of DPH decreased the exploratory tendency of the larvae and thus with DPH they swam in relatively limited areas, for example, swam in the areas along the wall and avoided the center of the test apparatus. However, in the current study, this explaration is not supported by the finding that under dark condition, there was no difference in the degree of anxiety between 20 µM DPH-treated groups and corresponding control groups, while the locomotor activity of 20 µM DPH-treated groups was significantly higher than that of control groups (Fig. 5&6). In addition, our results of correlation analysis between the locomotor activity and thigmotaxis in the DPH-treated groups, the level of anxiety of which groups was significantly lower than control groups, and corresponding control groups showed that there was no correlation between the locomotor activity and the degree of anxiety (Fig. 8), similar to the data reported before (Levin, Bencan et al. 2007; Li, Lin et al. 2014). g-Aminobutyric acid (GABA) is the principal inhibitory neurotransmitter of the brain and is one of several neurotransmitters that appear to be involved in the pathogenesis of anxiety. Drugs that stimulate GABA-A receptors, such as benzodiazepines (BDZs) and barbiturates, have anxiolytic and anti-seizure effects, whereas blocking the GABA-A receptor with antagonists leads to severe anxiety in man and in animals (Rodin and Calhoun 1970; File and Lister

ACCEPTED MANUSCRIPT 1984; Malizia, Cunningham et al. 1998). As a result, we can explore the mechanism of the anxiolytic effect of DPH by measuring the change in GABA. As previously reported (Emran, Rihel et al. 2008; MacPhail, Brooks et al. 2009), sudden illumination transition from light to dark introduced a dramatic increase in the locomotor activity of

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zebrafish larvae, while 5-dpf larvae that were treated with DPH still showed increased locomotor activity when given illumination challenge (Fig. 7), implying that DPH was not involved in the

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regulation of the stress response. Further work needs to be conducted to test this hypothesis. Additionally, our simultaneous monitoring study suggested that after the robust increases in the

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locomotor activity, larval zebrafish would maintain a high locomotor activity during the entire short dark phase and return to baseline locomotor activity immediately when receiving a sudden illumination transition from dark to light. However, a previous study found that the increased locomotor activity during the dark phase will gradually return to baseline locomotor activity (Emran, Rihel et al. 2008). It

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is possible that the duration of the dark period in our study was too short to observe the gradual course. Conclusion

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The present study aims to evaluate the effects of DPH on the locomotor activity and thigmotaxis of zebrafish larvae. The behavioral analysis showed that high doses of DPH inhibited the locomotor activity of zebrafish larvae, while low doses of DPH stimulated it. DPH possessed anxiolytic properties at high concentrations. The above effects were not affected by the light condition (light or dark). Visual

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stimuli (illumination challenge from light to dark) introduced a robust increase in the locomotor

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activity, while the increase was not eliminated by DPH treatment. In conclusion, the neurobehavioral effect of DPH was concentration-dependent. Acknowledgments

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This work was supported by National Natural Science Foundation of China Grant (81271509) and the Open Research Funds of the State Key Laboratory of Genetic Engineering of Fudan University to

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Li Q. Reference

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Figure legends:

ACCEPTED MANUSCRIPT Figure 1. Experimental procedure. (A) The experimental procedure was performed in four steps, including acclimatization (minutes 0–10 min), light phase (minutes 10-40 min), sudden illumination transition from light to dark and dark phase (minutes 40-45 min). (B) The testing apparatuses consist of each well in a 24-well plate format (diameter of 17 mm). The inner and outer zones were delineated as

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shown above. The width of the outer zone was set at 2.5 mm relative to the border of the well.

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Figure 2. Distance moved by larvae within the entire zone during 35-min measurements period. The distance moved in each 1-min time bin during 35-min measurements period (10-min to 45-min) were

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plotted against the progression of the experiment. The control group (filled circles) was plotted with a single DPH concentration group (open circles) in each panel. The shaded part in each panel represents the 5-min dark phase, and the non-shaded part in each panel represents the 30-min light phase. The data

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are presented as the mean ± SEM, n=32 animals per group.

Figure 3. Effects of DPH exposure on the locomotor activity of zebrafish larvae under light condition. The average distance moved by zebrafish larvae at 5-dpf within each 1-minute time bin during the

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30-min light phase were plotted. The data are presented as the mean ± SEM, n=32 animals per group. Statistical icons: *

Effects of diphenylhydantoin on locomotion and thigmotaxis of larval zebrafish.

Antiepileptic effects of diphenylhydantoin (DPH) have been documented in animal studies and clinical research, while little is known about the effects...
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