Locomotor activity assay in zebrafish larvae: Influence of age, strain and ethanol

Neurotoxicology and Teratology 34 (2012) 425–433

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Locomotor activity assay in zebrafish larvae: Influence of age, strain and ethanol☆ Celine de Esch a, 1, Herma van der Linde b, Roderick Slieker a, 2, Rob Willemsen b, André Wolterbeek a, Ruud Woutersen a, Didima De Groot a,⁎ a b

TNO, Research group Quality and Safety, Zeist, The Netherlands Erasmus MC, Department of Clinical Genetics, Rotterdam, The Netherlands

a r t i c l e

i n f o

Article history: Received 4 April 2011 Received in revised form 16 February 2012 Accepted 19 March 2012 Available online 29 March 2012 Keywords: Zebrafish larvae Strain Age Ethanol Light/dark Motor activity

a b s t r a c t Several characteristics warrant the zebrafish a refining animal model for toxicity testing in rodents, thereby contributing to the 3R principles (Replacement, Reduction, and Refinement) in animal testing, e.g. its small size, ease of obtaining a high number of progeny, external fertilization, transparency and rapid development of the embryo, and a basic understanding of its gene function and physiology. In this context we explored the motor activity pattern of zebrafish larvae, using a 96-well microtiter plate and a video-tracking system. Effects of induced light and darkness on locomotion of zebrafish larvae of different wild-type strains and ages (AB and TL, 5, 6 and 7 dpf; n = 25/group) were studied. Locomotion was also measured in zebrafish larvae after exposure to different concentrations of ethanol (0; 0.5; 1; 2 and 4%) (AB and TL strain, 6 dpf; n = 19/ group). Zebrafish larvae showed a relatively high swimming activity in darkness when compared to the activity in light. Small differences were found between wild-type strains and/or age. Ethanol exposure resulted in hyperactivity (0.5–2%) and in hypo-activity (4%). In addition, the limitations and/or relevance of the parameters distance moved, duration of movements and velocity are exemplified and discussed. Together, the results support the suggestion that zebrafish may act as an animal refining alternative for toxicity testing in rodents provided internal and external environmental stimuli are controlled. As such, light, age and strain differences must be taken into account. © 2012 Elsevier Inc. All rights reserved.

1. Introduction One of the most popular and best described vertebrate model species in developmental biology is the zebrafish (Danio rerio) (Barman, 1991; Bhat, 2003; Laale, 1977; Talwar and Jhingran, 1991). This freshwater fish offers a number of advantages in biomedical research including small size, low husbandry costs and easy maintenance. Zebrafish also allow the collection of high numbers of progeny all at once while the embryos develop rapidly; embryogenesis and organogenesis are completed within the first few days (Kimmel et al., 1995). Moreover, fertilization and development occur externally, permitting direct observation and manipulation under controlled conditions. In addition, the inherent transparency of the developing zebrafish embryo allows easy developmental staging combined with functional ☆ This research has been funded in part by The Netherlands' Ministry of Health, Welfare and Sports, the Ministry of Social Affairs and Employment, and by The Netherlands' Ministry of Defense under R&T Program V936 ‘Military Toxicology’. ⁎ Corresponding author at: Research Group Quality and Safety, TNO, P.O. Box 360, 3700 AJ Zeist, The Netherlands. Tel.: + 31 88 86 65 144; fax: + 31 30 69 44 954. E-mail address: [email protected] (D. De Groot). 1 Present address: Erasmus MC, Department of Clinical Genetics, Rotterdam, The Netherlands. 2 Present address: Leiden University MC, Molecular Epidemiology section, Leiden, The Netherlands. 0892-0362/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.ntt.2012.03.002

and morphological assessments (Chen et al., 1996; Fraysse et al., 2006; Samson et al., 2001). By now, the zebrafish genome has been sequenced and different genetic tools have been developed (Driever et al., 1996; Golling et al., 2002; Grunwald and Eisen, 2002; Knapik, 2000; Nasevicius and Ekker, 2000). Moreover, certain stereotypic behaviour of the zebrafish is well described and behavioural tests have been developed to assess effects on sensory, motor and cognitive behaviour (Gerlai, 2003; Miklosi and Andrew, 2006; Parng, 2005; Alderton et al., 2008). Recent studies with known mammalian neurotoxic and cardiotoxic agents have shown that these substances caused similar effects in zebrafish (Tillitt and Papoulias, 2002; Hen Chow and Cheng, 2003; Ton et al., 2006; Hill et al., 2003; Kari et al., 2007; Levin et al., 2003). These features make the zebrafish an excellent model organism to investigate toxicity. Zebrafish embryos and larvae are especially suitable for (drug induced) toxicity screening purposes since they can live in small volumes, for example in a 384-wells plate, for a few days. Hydrophobic compounds can permeate through their skin while hydrophilic compounds or large molecules or proteins can be injected into the yolk sac, or later the sinus venosus or circulation (Summerton and Weller, 1997; Milan et al., 2003; Fei et al., 2010). From 72 h post fertilisation (hpf) the larvae start to swallow and compounds can be administered orally as well (McGrath and Li, 2008). By 5 to 6 days post fertilisation, zebrafish larvae have developed distinct organs and tissues. Although zebrafish lack

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some of the mammalian organs, i.e. lung, prostate, and mammary glands, their organs and tissues are largely similar to their mammalian counterparts at the anatomical, physiological and molecular level (Wilson et al., 2002; Lewis and Eisen, 2003; Moens and Prince, 2002). Much is known about the behaviour of the adult zebrafish; however, the knowledge of zebrafish larvae is limited. Recent research has demonstrated that zebrafish larvae are subject to many intrinsic and extrinsic stimuli (MacPhail et al., 2009; Tierney, 2010). It should be borne in mind that the behaviour of zebrafish is susceptible to many dynamic processes that are not limited to minutes (habituation) or hours (time of the day), but have effects up to months (circadian rhythms/season) (MacPhail et al., 2009; Burgess and Granato, 2007). Other (environmental) factors might influence behaviour as well, including age (Padilla et al., 2011), strain (Loucks and Carvan III, 2004), size of well (Padilla et al., 2011), light conditions and water conditions. Regarding light conditions, alternating dark and light periods have been shown to influence the distance moved: in light periods the distance moved is relatively low and when the light is turned off the distance moved increases strongly (Padilla et al., 2011; Emran et al., 2007; Fischer et al., 1998). The zebrafish model needs to be carefully validated in the context of toxicology and drug discovery before it can be used for hazard identification and risk assessment. In this study we used the model compound ethanol which has extensively been studied in several species. Waterborne ethanol exposure has been shown to cause teratogenic responses and moreover ethanol exposure to zebrafish embryos causes craniofacial anomalies, developmental retardation, branchial skeleton defects, pericardial and yolk-sac oedema and increased mortality(Arenzana et al., 2006; Bilotta et al., 2004; Carvan III et al., 2004; Reimers et al., 2004; Bilotta et al., 2002; Blader and Strähle, 1998; Loucks and Carvan III, 2004). In the research described here, a behavioural assay was carried out with zebrafish larvae to study locomotor activity in different strains, under different lighting conditions, at different ages and after ethanol exposure. The commonly used parameters to assess the activity of zebrafish, i.e. distance moved, duration of movements and velocity of movements, were assessed and discussed. 2. Materials and methods 2.1. Animal welfare Animal welfare was maintained in accordance with the principles governing the use of animals in experiments of the European Communities (Directive 86/609/EEC) and Dutch Legislation (The Experiments on Animals Act. 1997). This included approval of the study by TNO's ethical review committee. Although zebrafish embryos up to 120 h post fertilization are not considered as experimental animals under European legislation, experiments with zebrafish younger than 120 hpf were also reported to TNO's ethical review committee 2.2. Housing Adult zebrafish (Danio rerio) kept within the TNO animal facility are offspring of Tupfel long fin (TL) and AB wild-type parental fish originally obtained from the Zebrafish International Resource Center (ZIRC). Zebrafish were held in colonies in self-regulating aquaria (Tecniplast, Tecnilab-BMI, The Netherlands) under standard conditions in aquarium water, defined as reverse osmosis water with a controlled pH, water temperature and conductivity of 7.5, 28 °C and 500 mS, respectively. Lighting was artificial with a sequence of 12 h light (7 AM to 7 PM) and 12 h darkness. To obtain zebrafish larvae adult couples, consisting of two male and one female fish, were placed in a breeding tank containing a partition (Tecniplast) in the afternoon. The fish were left undisturbed overnight and the next morning the partition was removed within half an hour after the onset of light. As soon as embryos were present, the adults were returned to their colony tank and all embryos from the same strain were pooled from the

breeding tanks and maintained at 28 °C on a 12:12 h light/dark cycle in plastic Petri dishes. The eggs were kept in aquarium water with 0.05% methylene blue to prevent fungal contamination for 24 h. The water was replaced with fresh aquarium water (still containing methylene blue up to day 4) on day 1, 3, 4 and 5 thereafter, while dead eggs and larvae were removed at the same time. After 5 days, the larvae were transferred to small tanks containing aquarium water (without methylene blue) and maintained at 28 °C. Feeding with Paramecia started at 5 days post fertilisation (dpf) and took place 4 times a day. 2.3. Motor Activity testing and experimental groups Motor activity assays were carried out using a ViewPoint behaviour recording system including the accompanying movement tracking and analysis software (Zebrabox, ViewPoint, France; tracking rate: 25 samples per second). This closed system consists of a camera placed above a chamber filled with circulating water and a temperature sensor. A microtiter plate is placed in a chamber which can then be illuminated with infrared and/or white lights using the software. After transferring the larvae individually with a plastic pipette, fish movements were measured in squared 96-wells microtiter plates (Whatman cat. No. 7701-1651) in a volume of ±500 μl under the following lighting conditions: 15 min of darkness (i.e. infrared light), 15 min of bright light and a final 15-minute period of darkness (further called ‘sessions’: DarkI, Light, DarkII). Light intensity was measured using a Voltcraft MS-1300 Lux meter (Voltcraft, Hirschau, Germany). The light intensity in the laboratory housing with the self-regulating aquaria was ±350 lx. To measure the intensity in the measured locomotor apparatus, the spherical sensor was placed on the transparent bottom of the chamber and the lid of the Zebrabox was closed. The light, turned on at the same intensity as used during the experiments, resulted in a measured light intensity of 700 lx, while the infrared light resulted in a measured intensity of 0 lx. All assays were executed in the afternoon (>2 PM) to ensure steady activity of the zebrafish (MacPhail et al., 2009), and water temperature was held at a constant temperature of 28 °C during the sessions. Prior to measurements the larvae were allowed to acclimatize for a period of 15 min in darkness. Larvae of both wild-type strains (AB and TL) aged 5, 6 or 7 days were tested in groups of 25 larvae each. The six groups were tested at the same day; the larvae were equally distributed over two plates in a randomly designed fashion and the two plates were tested subsequently. In addition, the same protocol was used to investigate the effects of ethanol: a drug with well-known effects on locomotor activity in humans and mammals. For this test, 6 day old larvae (TL and AB, n = 19/group) were exposed to 0, 0.5, 1, 2 or 4% ethanol (v/v, 100% pure, Sigma-Aldrich) dissolved in aquarium water. The different groups of fish were equally distributed over two 96-wells plates containing the appropriate solutions and these plates were analysed consecutively. Ethanol exposure took place from 30 min before the start of the motor activity assay until the end of the assay. Locomotor activity of zebrafish larvae expressed as Distance Moved (DM) per unit time was studied in both TL and AB zebrafish strains and the effects of different lighting conditions and different ages were analysed. Also the effect of exposure to various doses of ethanol on DM of 6 day old larvae was studied in both TL and AB strain. Here, also effects on the parameters Duration of Movements (DoM) per unit time and the (calculated) parameter Velocity of Movements (DoM) were studied; the three parameters were mutually compared to assess their relevance in toxicity testing. 2.4. Motor activity analysis and statistics Using the analysis software (Zebrabox), tracks were analyzed per animal for Distance Moved (DM (mm)) per unit time (1-minute or 5minute intervals per 15-minute session) as a measure for locomotor

C. de Esch et al. / Neurotoxicology and Teratology 34 (2012) 425–433

activity. In addition, Duration of Movement (seconds (s)) was expressed per 1-minute intervals. Mean Velocity of Movement was calculated ({Σ i = 1-n [Total Distancei/timei]}/n) by means of Windows Excel, and expressed in mm per second (mm/s) and displayed in 1-minute intervals. The values of 1-minute intervals were used for statistical comparison within and between age groups of both TL and AB strain for the different sessions DarkI, Light and DarkII. To study differences between sessions within an age group, differences between age groups per session, and interaction between time and group, a Repeated Measures ANOVA procedure was conducted on the dataset followed by post-hoc comparison (Bonferroni or Dunnett, p b 0.05 significant) (SAS, version 8.2e, USA). The values of 5-minute intervals per session were used to study more precisely immediate effects of a sudden change in lighting condition, such as occurs at the session interchange from the DarkI to the Light session and from the Light to the DarkII session, as these effects may differ in zebrafish larvae of different age and strain. The choice to study 5-minute intervals for this analysis was based on visual interpretation of the 1-minute interval Distance Moved graphs considering that the fluctuations observable in 1-minute intervals near the session interchange could lead to false conclusions. So first, the distance moved was expressed per 5 min intervals and compared between the age groups for each of the four intervals near the session change from DarkI to Light (interval 11–15 min; interval 16–20 min) and from Light to DarkII (interval 26–30 min; interval 31–35 min); second, the group means of the differences between the adjacent 5 min intervals from DarkI to Light, and from Light to DarkII were compared (ANOVA/ Bonferroni or Dunnett, p b 0.05, significant). Results of these statistical comparisons are summarized in Tables.

3. Results 3.1. Motor activity zebrafish: Effects of lighting conditions, strain and age 3.1.1. Session means of Distance Moved (mm/min), Duration of Movements (s/min) and Velocity of Movements (mm/s) Effects of age and strain on swimming activity of zebrafish larvae expressed in session means of the parameters Distance Moved (mm/min), Duration of Movements (s/min) and Velocity of Movements (mm/s) are shown in Table 1. For AB and TL wild-type strains of 5, 6 and 7 dpf, the following general motor activity pattern was observed. In the first period of darkness (DarkI), the average Distance Moved per 15 min session was relatively high (about 95 mm or more), while the average distance moved decreased in the light period (Light) (to about 80 mm or less). In the second dark period (DarkII), activity increased again to average values above ± 75 mm. The average Duration of Movements measured per session was ≥20s/min in Dark I, ≤20 s/min in Light and ultimately ≥20s/min in Dark II; the average Velocity measured was ≥4.0 mm/s (Dark I), ≤4.0 mm/s (Light) and ≥4.0 mm/s (Dark II). As is shown in the table, small differences between larvae of the AB and TL wild-type strains were visible. However, inter-individual differences in Velocity over time are known to occur since Velocity is affected by factors like animal size and well size, and the Velocity indirectly affects the Distance Moved and Duration of Movements (refer e.g. (Plaut, 2000)). So, a more detailed insight into the swimming curves is required to further pinpoint differences. Therefore, Distance Moved per 1- and 5-minute intervals is further discussed.

3.1.2. One-minute interval means of Distance Moved: swimming pattern over time The Distance Moved per 1-minute interval for 5, 6 and 7 day old larvae over the 15-minute periods of alternating darkness and light is illustrated in Fig. 1.

427

Table 1 Effects of age and strain on swimming activity of zebrafish larvae. Session means of the different parameters for zebrafish larvae of different age (5, 6 or 7 days post fertilization (dpf)) in both AB and TL wild-type strains. Parameter

Distance Moved (mm/min)

Strain

AB

TL

Duration of Movements (s/min)

AB

TL

Velocity of Movements (mm/s)

AB

TL

Age (dpf) 5 6 7 5 6 7 5 6 7 5 6 7 5 6 7 5 6 7

DarkI 1) 2

Light 1) 2

DarkII 1)

Mean )

SEM

Mean )

SEM

Mean2)

SEM

152.4 125.8 96.1 104.6 98.1 132.5 25.6 31.0 20.9 20.5 19.8 25.3 5.79 4.36 4.41 5.01 4.65 4.98

4.08 3.21 3.23 4.17 3.81 3.57 4.08 3.21 3.23 4.17 3.81 3.57 4.08 3.21 3.23 4.17 3.81 3.57

27.6 43.5 70.0 24.8 43.3 78.1 6.8 15.4 17.6 8.4 11.5 18.4 3.81 3.17 3.93 3.35 3.70 4.07

2.49 2.14 2.81 1.98 2.47 2.89 2.49 2.14 2.81 1.98 2.47 2.89 2.49 2.14 2.81 1.98 2.47 2.89

119.2 116.7 117.4 76.3 90.1 127.6 20.3 29.1 24.5 18.9 19.1 25.4 5.29 4.13 4.46 4.16 4.23 4.60

5.21 4.14 4.24 4.04 4.01 4.20 5.21 4.14 4.24 4.04 4.01 4.20 5.21 4.14 4.24 4.04 4.01 4.20

1) Duration of each session (DarkI, Light and DarkII) is 15 min. 2) Number of fish larvae: n = 25.

3.1.2.1. AB-strain. In DarkI, the Distance Moved (DM) per minute (Fig. 1A) started relatively high (>100 mm/min; DM AB 5 dpf > 6 dpf > 7 dpf), followed after about 3 min by a gradual decrease over time for all 3 age groups (DM AB 5 dpf > 6 dpf > 7 dpf). In the Light period, when the light was turned on after 15 min, the activity of the 6 and especially the 5 day old larvae decreased further (DM AB 7 dpf > 6 dpf > 5 dpf) and remained relatively low in all age groups (b80 mm/min) until the light was turned back off. In DarkII, immediately after turning off the light, distance moved by the larvae peaked before gradually decreasing again. No clear differences between the age groups were observed in DarkII. Comparing the curves in DarkII with those in DarkI, the slope of the curve in the DarkII period appeared steeper at first sight for all age groups compared to the curve in DarkI. Statistical analysis of the effects observed in wild-type AB larvae demonstrated that the differences over time within an age group were significant (Repeated Measures ANOVA: F(2,3231) = 416.98, p b 0.0001), but not those observed between age groups. However, the interaction between the age of the zebrafish and the session was significant (F(4,3231) = 46.12, p b 0.0001). In fact, the Distance Moved in the Light session (Bonferroni posthoc analysis) differed significantly from both DarkI (pb 0.0001) and DarkII sessions (pb 0.0001); the difference between DarkI and DarkII, in turn, showed a trend (0.1 > p > 0.05). Compared to the activity of the AB 6 dpf and 7 dpf, the activity of AB 5 dpf larvae was the highest in the Dark periods (DarkI> DarkII) and the lowest in the Light period. However, the differences between the age groups did not reach significance. 3.1.2.2. TL-strain. The overall pattern of motor activity or Distance Moved of TL wild-type larvae (Fig. 1B) was rather similar to that of the AB-larvae. In addition, one should notice that for both AB- and TL strains the gradual decrease in activity (Distance Moved/min) observed in the first dark period (DarkI) was preceded by a short gradual increase in activity during the first three minutes in the 5 and 6 day larvae but not in the 7 day old larvae. As for the AB wild-type strain, the TL larvae also showed a slightly steeper slope in the DarkII period compared to the DarkI period at first sight. Statistical comparison (Repeated Measures ANOVA, p b 0.05 significant) of the Distance Moved of TL wild-type larvae within and between age groups over the different sessions pointed at significant differences between sessions (F(2,3321) = 261.63; p b 0.0001), significant age effects between and

C. de Esch et al. / Neurotoxicology and Teratology 34 (2012) 425–433

Distance Moved (mm)

A 300

B 300

AB wildtype larvae: Distance Moved AB 5 dpf AB 6 dpf AB 7 dpf

200

100

Distance Moved (mm)

428

TL wildtype larvae: Distance Moved TL 5 dpf TL 6 dpf TL 7 dpf

200

100

0

0 0

5

10

15

20

25

30

35

40

45

0

5

10

Time (min) DarkI

Light

15

20

25

30

35

40

45

Time (min) DarkII

DarkI

Light

DarkII

Fig. 1. Effects of age and strain on swimming activity of zebrafish larvae. Distance Moved (± SEM) per 1-minute intervals for AB wild type (A) and TL wild type (B) larvae of 5, 6 and 7 days post fertilization (dpf), in alternating light and dark periods. In DarkI, a gradual decrease in activity is observed after an initial increase of about 3 min for the 5 and 6 dpf larvae of both wild types. Activity is low in the Light session. In DarkII, immediately when the light is turned off, activity peaks. Notice that these effects are most pronounced in the 5 dpf larvae of the AB wild type.Statistical key: Repeated Measures ANOVA, p b 0.05, significant: A) AB wild type: significant differences between sessions (F(2,3231) = 416.98; p b 0.0001), age effects (F(2,3231) = 1.79, p = 0.1676), and interaction between session and age (F(4,3231) = 46.12; p b 0.0001) are observed. B) TL wild type: significant differences between sessions (F(2,3321) = 261.63; p b 0.0001), significant age effects (F(2,3321) = 131.90; p b 0.0001) and significant interaction between session and age (F(4,3321) = 5.05; p = 0.0005) are observed.

within groups (F(2,3321) = 131.90; p b 0.0001), and a significant interaction between session and age (F(4,3321) = 5.05; p = 0.0005). Post-hoc comparison (Bonferroni, p b 0.05 significant) confirmed that these differences resembled those observed for AB wild-type larvae, except that in TL wild-type larvae all session and group comparisons reached the level of significance (p b 0.05). 3.1.3. Five-minute interval means: Effects of ‘sudden’ change in lighting at the session interchange The 5-minute intervals per session were used to study statistically more precise the intervals near the interchange of the Dark I to Light and Light to Dark II sessions to study a sudden change in lighting. The Distance Moved displayed per 5-min intervals is shown for the 5, 6 and 7 dpf larvae of the AB (Fig. 2A) and the TL strain (Fig. 2B). Comparison of the Distance Moved per 5-minute interval for the four intervals near the session interchanges revealed the following differences (ANOVA /Bonferroni, p b 0.05, significant) [cf Table 2]. In both strains, the 5 day old larvae showed a significantly higher DM in the last DarkI interval (11–15 min) compared to the larvae of 7 dpf. However, the 7 dpf larvae of both strains showed a significantly higher DM in the first five minutes of light. In the last five minutes of light (26–30 min) the 7 dpf AB larvae displayed a significantly higher

Total Distance Moved

B

AB wildtype per 5 minutes

5 dpf

Distance Moved (mm)

300

6 dpf

250

7 dpf 200 150 100 50 0

Total Distance Moved TL wildtype per 5 minutes

300

Distance Moved (mm)

A

DM only compared to the 5 dpf while in the TL strain the DM of 7 dpf larvae was significantly higher compared to both other age groups. In the first interval of DarkII the only significant difference was found in TL larvae (DM 7 dpf > DM 6 dpf). The individual difference in Distance Moved between the two adjacent intervals from DarkI to Light (intervals: 11–15 min; 15–20 min) and from Light to DarkII (intervals: 26–30 min; 31–35 min) were calculated, averaged per age group (mm ± SEM) and compared between groups (ANOVA/Bonferroni, p b 0.05, significant). Results of this statistical comparison demonstrated [cf Table 3] that the largest significant effect of a sudden change in lighting condition occurred at the interchange from Light to the DarkII for all three age groups of both wild-type strains AB and TL [compare Figs. 1 and 2, p b 0.0001]. The changes from DarkI to Light were mild, and did not reach significance for the TL wild-type 5, 6 and 7 dpf larvae. The 5 and 6 dpf AB-larvae clearly deviated from other age groups [compare also Fig. 2A]: these larvae clearly showed a larger reaction and change in activity when the light was turned on (from DarkI to Light session (respectively p b 0.0001 or p = 0.007). The results on effects of lighting on Distance Moved –taken together– demonstrate [compare Figs. 1 and 2] that an age related effect existed (activity of 7 dpf larvae> 6 dpf >5 dpf) in the 5-minute light intervals

5 dpf 6 dpf

250

7 dpf 200 150 100 50 0

0-5

6-10 11-15 16-20 21-25 26-30 31-35 36-40 41-45

0-5

6-10 11-15 16-20 21-25 26-30 31-35 36-40 41-45

Period of test (min)

DarkI

Light

Period of test (min)

DarkII

DarkI

Light

DarkII

Fig. 2. Effects of age and strain on swimming activity of zebrafish larvae during “sudden” lighting changes at the interchange between the sessions; AB wild-type (A) and the TL wild-type larvae (B) of 5, 6 and 7 days post fertilization (dpf).The Distance Moved is expressed per 5-minute intervals and compared between the age groups for each of the four intervals near the session interchanges from DarkI to Light (interval 11–15 min; interval 16–20 min) and from Light to DarkII (interval 26–30 min; interval 31–35 min); furthermore, the group means of the differences between the adjacent 5-minute intervals from DarkI to Light, and from Light to DarkII are compared (ANOVA/Bonferroni, p b 0.05, significant). Results of these statistical comparisons are summarized in Tables 2 and 3 under the Results section.

C. de Esch et al. / Neurotoxicology and Teratology 34 (2012) 425–433 Table 2 Distance Moved per 5-minute intervals near the session interchange. Statistical comparison of group means per interval: DarkI (3rd Interval 11–15 min), Light (1st Interval 16–20 min session), Light (3rd Interval 26–30 min), DarkII session (1st Interval 31–35). Statistical key: ANOVA/Bonferroni, p b 0.05, significant. Session/interval

Strain

Statistically significant effects between age groups (dpf*)

DarkI session, 3rd interval (11–15 min)

AB TL AB TL AB TL AB TL

7 dpf b 5 dpf 7 dpf > 5 dpf 7 dpf > 6 dpf; 7 dpf > 5 dpf 7 dpf > 6 dpf; 7 dpf >5 dpf 7 dpf > 5 dpf 7 dpf > 6 dpf; 7 dpf > 5 dpf Not significant 7 dpf > 6 dpf

Light session, 1st Interval (16–20 min) Light session, 3rd Interval (26–30 min) DarkII session, 1st Interval (31–35 min) * dpf, days post fertilization.

of both wild-type strains (AB and TL). Strikingly, whereas this holds also true for the tested 5-minute dark intervals of the TL wild-type larvae, for the AB wild-type larvae, deviating behaviour is observed particularly for the 5 dpf larvae. These 5 dpf AB wild-type larvae clearly show a larger reaction and change in activity when the light is turned off (from DarkI to Light session) and also from Light to DarkII session. 3.2. Ethanol exposure of AB and TL wild-type strains; 6 days post fertilization (dpf) 3.2.1. One- and 5-minute intervals of Distance Moved by AB and TL wild-type strains, 6 days post fertilization (dpf): effects of ethanol during different lighting conditions Figs. 3 and 4 display the Distance Moved of both wild-type strains, respectively AB and TL, under the different lighting conditions after ethanol exposure (0%, 0.5%, 1%, 2% or 4% vv), expressed per 1-minute intervals (Figs. 3A and 4A) and per 5-minute intervals (Figs. 3B and 4B). 3.2.1.1. AB-strain. The general activity pattern of the control (0% ethanol) group of the AB wild-type showed a slightly decreased activity in Light compared to the activity in both dark periods, while the swimming activity in DarkI was lower compared to the average activity in DarkII. The same general pattern was seen in ethanol exposed larvae except for the 4% dose group, which showed a very low Distance Moved throughout all the sessions. Statistical comparison (Repeated Measures ANOVA, p b 0.05 significant) of the Distance Moved within and between groups over the different sessions pointed at significant dose effects (F(4,2958) = 803.17, p b 0.0001), significant differences between sessions (F(2,2958) = 101.49, p b 0.0001) and significant interactions between dose and session (F(8,2958) = 22.73 p b 0.0001). Activity clearly differed between sessions within the groups and different activity was observed between groups (Dunnett post-hoc comparison, p b 0.05, significant), i.e. activity in the 1% ethanol dose group was higher than the control group in all three sessions, whereas the

Table 3 Results of statistical comparison of activity changes per 5-minute intervals at the interchange of the sessions from DarkI (3rd Interval 11–15 min) to Light (1st Interval 16– 20 min session) and from Light (3rd Interval 26–30 min) to DarkII session (1st Interval 31–35). Statistical key: ANOVA /Bonferroni, p2 b 0.05, significant. Session Interchange

Age (dpf*)

AB Wild-type

TL Wild-type

DarkI to Light DarkI to Light DarkI to Light Light to Dark II Light to Dark II Light to Dark II

5 6 7 5 6 7

b0.0001 0.007 0.742 n.s. b0.0001 b0.0001 b0.0001

0.4956 n.s. 1.0000 n.s. 0.1918 n.s. b 0.0001 b 0.0001 b 0.0001

*dpf, days post fertilization.

429

activity in the 4% dose group was significantly lower. The activity of the 0.5% dose group showed a trend (0.1 > p > 0.05) towards increased activity at the beginning of the DarkI (about 3 min) and DarkII sessions (about 6 min); activity of the 2% dose group was significantly reduced during the DarkII session compared to the control group. 3.2.1.2. TL-strain. The activity pattern of the TL larvae resembled that of the AB larvae (cf. Figs. 4A and 3A) although effects and changes were less pronounced. Statistical comparison (Repeated Measures ANOVA, p b 0.05 significant) of the Distance Moved within and between dose groups pointed at significant session effects (F(2,2832) = 52.34 p b .0001), significant dose effects (F(4,2832) = 593.93, p b .0001) and a significant interaction between session and dose (F(8,2832) = 8.15, p b .0001). Ethanol induced a mild increase in activity in the 1% dose group and clear hypo-activity in the 4% ethanol group (Dunnett post-hoc comparison, p b 0.0001). The changes in activity observed in the AB wild-type strain during the first interval of the DarkII session right after the light was turned off again, can hardly – if at all – be observed in the TL wild-type strain [see below: changes at the session interchange]. 3.2.1.3. Ethanol exposure and activity changes at the interchange of Dark I to Light and Light to Dark II sessions. The values of 5-minute intervals per session, used to study the intervals near a session interchange for effects of a sudden change in lightning as occurs when the light is switched on or off is shown in Figs. 3B and 4B for respectively AB and TL wild-type larvae of 6 days post fertilization (dpf). Statistical comparison of the Distance Moved group means per 5-minute interval for the four intervals near the session changes, revealed the following significant differences (Statistical key: ANOVA /Bonferroni, p b 0.05, significant) [Table 4]. The individual difference in Distance Moved between the two adjacent intervals from DarkI to Light (intervals: 11–15 min; 15–20 min) and from Light to DarkII (intervals: 26–30 min; 31–35 min) were calculated, averaged per age group (mm ± SEM) and compared between groups (ANOVA/Bonferroni, p b 0.05, significant). Results of this statistical comparison are shown in Table 5, demonstrating that the largest effect of a sudden change in lighting condition on Distance Moved occurred at the interchange from Light to the DarkII which appeared significant for the 0% ethanol control group, the 0.5% and 1% groups of AB wild-type strain (6 dpf larvae) and the 0.5% group of TL wild-type strain (6 dpf larvae). The changes from DarkI to Light were mild, and did not reach significance. The results on effects of ethanol and lighting on Distance Moved – taken together – demonstrated [compare also Figs. 3 and 4] that a response was observed when the light is switched off in the control (0%) and 0.5% ethanol dose groups of both wild-type strains AB and TL; also dose group 1% of the AB wild-type strain reacts similar to the control group. However, at higher dose groups this ‘startle’ reaction was suppressed. 3.2.2. Ethanol exposure of AB wild-type strain; 6 days post fertilization (dpf). Additional value of Duration and Velocity of Movements Distance Moved, Duration of Movements and Velocity are commonly used measures to assess activity in zebrafish. Here we looked at all three measures to assess their value in translating zebrafish activity and this is illustrated in Fig. 5A (Duration of Movements, s/min) and 5B (Velocity of Movements, mm/s), taking the AB larvae exposed to ethanol as an example (compare with Fig. 3A: Distance Moved/min: mm). The Duration of movements (Fig. 5A) showed a similar pattern compared to the pattern found for Distance Moved (see Fig. 3A): a decreased activity in the light session with an increase in activity in the DarkII period. Statistical comparison of the Duration of Movements within and between groups over the different sessions pointed at significant dose effects (F(4,2977) = 974.13, p b 0.0001), significant session effects (F(2,2977) = 70.81, p b 0.0001) and a significant interaction

C. de Esch et al. / Neurotoxicology and Teratology 34 (2012) 425–433

A

Total Distance Moved

Total Distance Moved (mm)

AB wildtype per minute

0% ethanol

B

Total Distance Moved

0.5% ethanol 1.0% ethanol

400

2.0% ethanol

350

4.0% ethanol

300 250 200 150 100 50 0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45

AB wildtype per 5 minutes Total Distance Moved (mm)

430

Light

0.5% ethanol 1.0% ethanol

400

2.0% ethanol

350

4.0% ethanol

300 250 200 150 100 50 0 0-5

6-10 11-15 16-20 21-25 26-30 31-35 36-40 41-45

Period of test (min) DarkI

0% ethanol

Period of test (min) DarkII

DarkI

Light

DarkII

Fig. 3. Effects of ethanol exposure (0%, 0.5%, 1%, 2%, 4% v/v) on swimming activity in alternating light and dark periods of zebrafish larvae of AB wild-type strain, age 6 days post fertilization (dpf). A) Distance Moved (± SEM) per 1-minute. Exposure of 6 dpf AB wild-type larvae to ethanol has no effect on swimming activity of the 0.5% dose group during the first two sessions (DarkI and Light), but differs significantly from the control in the DarkII session. Induced hyperactivity in the 1% dose group and hypoactivity in the highest (4%) dose group is observed in all sessions. Moreover, in the DarkII session, the activity of the 2% group is decreased as well. Statistical key: Repeated Measures ANOVA/Dunnett posthoc comparison, p b 0.05, significant. Significant differences in sessions are found (F(2,2958) = 101.49; p b 0.0001), significant dose effects (F(4,2958) = 803.17; p b 0.0001), and significant interactions between dose and session (F(8,2958) = 22.73; p b 0.0001). B) Distance Moved (± SEM) per 5-minute intervals is used to study effects on swimming activity during “sudden” lighting changes at the interchange between the sessions. The Distance Moved is compared between the dose groups for each of the four intervals near the session change from DarkI to Light (interval 11–15 min; interval 16–20 min) and from Light to DarkII (interval 26–30 min; interval 31–35 min). Furthermore, the group means of the differences between the adjacent 5-minute intervals from DarkI to Light, and from Light to DarkII are compared (ANOVA/Bonferroni, p b 0.05, significant). Results of these statistical comparisons are summarized in Tables 4 and 5 under the Results section.

Total Distance Moved (mm)

A

0% ethanol

Total Distance Moved

0.5% ethanol

TL wildtype per minute

1.0% ethanol

400

2.0% ethanol

350

4.0% ethanol

300 250 200 150 100 50 0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45

(p = 0.0196). Strikingly, a highly significant increase in Velocity was observed in the 4% ethanol dose group, whereas this dose group showed the lowest Distance Moved and shortest Duration of Movements. Also the large variation among individuals within the 4% ethanol group should be noticed. Basically, the larvae exposed to 4% ethanol only produce very rapid and very short movements which – in the end – only result in a high swimming Velocity but not in a high Distance Moved. This leads to the conclusion that the measure Velocity might not be a good representative for general locomotor activity, and thus it is better to look at Distance Moved over time to compare motor activity of different groups of fish. 4. Discussion In this study, zebrafish locomotor activity or distance moved was easily and rapidly measured under different lighting conditions using an automated video image analysis system. Zebrafish larvae displayed

B Total Distance Moved Total Distance Moved (mm)

between dose and session (F(8,2977) = 21.93, p b 0.0001). This leads to the conclusion that total Duration of Movements significantly differs between the different ethanol-exposure groups and between the different sessions. Bonferroni posthoc comparison (pb 0.05, significant) showed that the Duration of Movements differed significantly between all three sessions (DarkII > DarkI> Light). The Duration of Movements of the 1, 2 and 4% ethanol groups differed significantly from the control group (1% > 0%; 2% b 0%, particularly in DarkII; 4%b 1%, over all sessions) while the Duration of Movements in the 0.5% ethanol group did not differ significantly from the control group. The Velocity of Movements on the other hand shows a different pattern compared to the Distance Moved and Duration of Movements (compare Fig. 5B with Figs. 3A and 5A). No differences in Velocity are observed between the three sessions DarkI, Light and DarkII. With regard to the ethanol dose groups, Bonferroni posthoc comparison (p b 0.05) showed that the 0.5% and 2% groups did not differ from the control group; the 1% group however reached significance

TL wildtype per 5 minutes 400

Light

0.5% ethanol 1.0% ethanol 2.0% ethanol 4.0% ethanol

300 200 100 0 0-5

6-10 11-15 16-20 21-25 26-30 31-35 36-40 41-45

Period of test (min) DarkI

0% ethanol

Period of test (min) DarkII

DarkI

Light

DarkII

Fig. 4. Effects of ethanol exposure (0%, 0.5%, 1%, 2%, 4% v/v) on swimming activity of zebrafish larvae of TL wildtype strain, age 6 days post fertilization (dpf). A) Distance Moved (± SEM) per 1-minute intervals in alternating light and dark periods. Notice that the activity pattern resembles that of the AB larvae alike [compare Fig. 3A], although the effects are less pronounced. Statistical comparison of the Distance Moved within and between dose groups points at significant session effects (F(2,2832) = 52.34 p b .0001), significant dose effects (F(4,2832) = 593.93, p b .0001) and significant interaction between dose and session (F(8,2832) = 8.15, p b .0001); Repeated Measures ANOVA/Dunnett, p b 0.05, significant. B) Distance Moved (± SEM) per 5-minute intervals to study effects on swimming activity during “sudden” lighting changes at the interchange between the sessions. The Distance Moved is compared between the dose groups for each of the four intervals near the session interchange from DarkI to Light (interval 11–15 min; interval 16–20 min) and from Light to DarkII (interval 26–30 min; interval 31–35 min). Furthermore, the group means of the differences between the adjacent 5-minute intervals from DarkI to Light, and from Light to DarkII are compared (ANOVA/Bonferroni, p b 0.05, significant). Results of these statistical comparisons are summarized in Tables 4 and 5 under the Results section.

C. de Esch et al. / Neurotoxicology and Teratology 34 (2012) 425–433 Table 4 Distance Moved per 5-minute intervals near the session interchange. Statistical comparison of groups means per interval: DarkI (3rd Interval 11–15 min), Light (1st Interval 16–20 min session), Light (3rd Interval 26–30 min), DarkII session (1st Interval 31–35). Statistical key: ANOVA /Bonferroni, p b 0.05, significant. Session/interval

Strain

Statistically significant effects between ethanol dose groups (%*)

DarkI session, 3rd interval (11–15 min)

AB

0% b 1%; 0% > 4%; 0.5% b 1%; 0.5% > 4%; 1% > 2%; 1% > 4%; 2% > 4% Not significant 0% b 1%; 0% > 4%; 0.5% b 1%; 0.5% > 4%; 1% > 4% 1% > 4%; 2% > 4% 0% b 1%; 0.5% b 1%; 0.5% > 4%; 1% > 2%; 1% > 4%, 2% > 4% 1% > 4%; 2% > 4% 0% > 4%; 0.5% > 4%; 1% > 2%; 1% > 4%; 2% > 4% 0% b 1%; 0% > 4%; 0.5% > 4%; 1% > 2%; 1% > 4%; 2% > 4%.

TL AB

Light session, 1st Interval (16–20 min)

TL AB

Light session, 3rd Interval (26–30 min)

TL AB

DarkII session, 1st Interval (31–35 min)

TL *Ethanol groups: 0, 0.5, 1, 2, 4%.

a distinct motor activity pattern under different lighting conditions. In light, zebrafish larvae showed a relatively low activity while in darkness a relatively high activity was observed. Transition periods showed sharp activity changes within a minute from the light change, especially from the light to the second darkness session. Recent studies with zebrafish larvae have found similar swimming activity patterns under alternating lighting conditions. (Burgess and Granato, 2007; Emran et al., 2008; Hurd et al., 1998; Prober et al., 2006) These studies also reported darkness (or infrared light) induced spikes in motor activity and gradually increasing motor activity during light periods. One explanation for the high activity in darkness might be in the observation that diurnal fish species aim to find shelter prior to night time and start to seek for cover as soon as night falls (Helfman, 1986). Another explanation would be that, in nature, fish will try to navigate away from debris in the water that occludes light. An increased activity in response to a predator in the water, which also occludes light, would be another explanation, but Burgess and Granato (Burgess and Granato, 2007) have shown that this is highly unlikely. They showed that the first responses of larvae were made towards the light occlusion (or predator) in contrast to escape trajectories in adult fish that navigate the fish away from the predator (Burgess and Granato, 2007). In a period of darkness, locomotor activity gradually declined while in light, locomotor activity gradually increased over time. During the different lighting periods of 15 min, locomotor activity did not reach a steady state. These response patterns may reflect habituation, an ancestral form of learning, but more research is needed to find a complete explanation (MacPhail

Table 5 Results of statistical comparison of activity changes per 5-minute intervals at the interchange of the sessions from DarkI (3rd Interval 11–15 min) to Light (1st Interval 16– 20 min session) and from Light (3rd Interval 26–30 min) to DarkII session (1st Interval 31–35). Statistical key: ANOVA/Dunnett, p b 0.05, significant. Session Interchange

Dose Group

AB Wild-type

TL Wild-type

DarkI to Light DarkI to Light DarkI to Light DarkI to Light DarkI to Light Light to Dark II Light to Dark II Light to Dark II Light to Dark II Light to Dark II

Ethanol 0% Ethanol 0.5% Ethanol 1.0% Ethanol 2.0% Ethanol 4.0% Ethanol 0% Ethanol 0.5% Ethanol 1.0% Ethanol 2.0% Ethanol 4.0%

0.9037 n.s. 1.0000 n.s. 1.0000 n.s. 1.0000 n.s. 1.0000 n.s. 0.0049 b0.0001 b0.0001 1.0000 n.s. 1.0000 n.s.

1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 0.0133 0.1491 1.0000 1.0000

n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.

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et al., 2009; Thompson and Spencer, 1966). It is noticed that in the research presented here, all assays were executed in the afternoon to ensure steady activity of the zebrafish (MacPhail et al., 2009). Age was found to have an influence on the motor activity pattern of zebrafish larvae. During the first days of life, larvae feed from their yolk sac, and in our laboratory exogenous feeding was started on day 5. As a result, the larvae of the different age groups have a slightly different feeding state. In addition, some small differences in body length and swim movements were observed in zebrafish larvae from the different age groups (unpublished observations), which might have had an influence on swimming activity as well. These findings are in agreement with literature published earlier (Padilla et al., 2011; Colwill and Creton, 2010). Strain also influenced motor activity. Five and six day old TL-larvae showed a lower average distance moved in all periods compared to the AB-larvae from the same age (only significant in both dark periods; Table 1). Seven day old TL-larvae however, showed a significantly higher activity than seven day old AB-larvae in light and darkness. Although background differences were found, similar reactions to a light change were observed in zebrafish larvae of different ages and strains. Larvae exposed to 4% ethanol produced very rapid and short movements resulting in high swimming velocity without an increase of the distance moved. Winter et al. (Winter et al., 2008) used the quantification of (high) speed locomotion as a classification method of seizure-like locomotor activity. Winters’ validation data suggests that the assay may offer a potential screening method in the early drug discovery pathway (Winter et al., 2008). However, to get a complete picture of the locomotor behaviour of zebrafish larvae, velocity needs to be analysed very carefully since the effects found on swimming velocity might not always completely represent the effects on distance moved, i.e. a higher average swimming velocity might not result in a higher average distance moved. Moreover, even when ‘high speed movement’ is observed one should be cautious to speak about seizure-like locomotor activity, as fast muscle contractions do not necessarily express brain seizure activity. Ethanol, a known drug of abuse with various effects on histological and behavioural level in humans, has been shown to increase as well as decrease motor activity in both humans and rodents. These effects on activity seem to depend on a number of experimental parameters, e.g. species and strain, dose of ethanol, time of consumption (Lewis and June, 1990; Tabakoff and Kiianmaa, 1982; Masur et al., 1986; Masur and dos Santos, 1988; Selderslaghs et al., 2010; Sylvain et al., 2010; Camarini et al., 2010; Jerlhag et al., 2010). The effects of ethanol on the swimming activity of zebrafish larvae have been investigated and it has been shown that exposure to low doses (0.5–2%) causes an increased swimming activity, while a decreased activity has been observed after exposure to high doses (4%) (MacPhail et al., 2009; Irons et al., 2009; Lockwood et al., 2004). Whereas in control animals a change in distance moved can be observed when the light is turned on (Fig. 1), ethanol exposed animals seem not to respond when the light is turned on. The results shown in the present study are in agreement with results shown before. Although these studies used different experimental systems, including Viewpoint (this study), Noldus Information Technology (MacPhail et al., 2009; Irons et al., 2009) and DIAS (Lockwood et al., 2004), the effect of different doses of ethanol were in agreement with each other. Therefore, the methods used can be considered robust for toxicity testing. It was found that under the circumstances of the present study, the reactivity to sudden light/dark changes was exacerbated by low dosages of ethanol (0.5 b 2%) and depressed by exposure to higher dosages (≥ 2% ethanol). The activity changes at the session-transitions – all or not under exposure to ethanol – were about the same for 6 dpf larvae of the AB and TL wild-type strains. Interestingly, the TL larvae showed the clearest dose response reaction to ethanol; hyperactivity was observed in all exposed groups except in the highest dose group which showed hypo-activity.

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C. de Esch et al. / Neurotoxicology and Teratology 34 (2012) 425–433

A

Total Duration of Movements AB wildtype per minute

0% ethanol

B

Total Velocity of Movements

0.5% ethanol

AB wildtype per minute

1.0% ethanol

50

2.0% ethanol 4.0% ethanol

40 30 20 10

0.5% ethanol 1.0% ethanol 2.0% ethanol 4.0% ethanol

Velocity (mm/s)

Total Duration (s)

50

0% ethanol

40 30 20 10 0

0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45

Period of test (min)

Period of test (min) DarkI

Light

DarkII

DarkI

Light

DarkII

Fig. 5. Ethanol exposure (0%, 0.5%, 1%, 2%, 4% v/v) on zebrafish larvae of AB wild-type strain, age 6 days post fertilization (dpf). A) Effects on total Duration of Movements (DoM) (s/min ± SEM) per 1-minute intervals. Notice how DoM follows the same pattern as Distance Moved [cf. Fig. 3A]. Statistical comparison points at significant differences between sessions (F(2,2977) = 70.81; p b 0.0001), significant dose effects (F(4,2977) = 974.13; p b 0.0001) and significant interactions between dose and session (F(8,2977) = 21.93; p b 0.0001). B) Effects on Velocity of Movements (VoM) (mm/s ± SEM) per 1-minute intervals. Since DM and DoM follow similar patterns over time[cf Figs. 3A, 5A, B], calculated Velocity per dose group is expected to reach a more or less constant level over all the sessions. This holds true here for the control and ethanol groups, except for the 4% ethanol group showing large variation in activity in addition. Statistical comparison points at significant dose effects (F(4,2273) = 47.15; p b 0.0001) and significant interactions between dose and session (F(8,2273) = 3.35; p = 0.0008). However, no significant differences between sessions (F(2,2273) = 2.03; p = 0.1319) exist.Statistical key: Repeated Measures ANOVA, p b 0.05, significant.

5. Conclusions In conclusion, swimming activity was shown to be influenced by lighting conditions, age, strain and ethanol exposure. Although background differences were detected, a highly reproducible, three-stage pattern of locomotor activity was established. These results show that locomotor activity can be reliably measured and quickly quantified in larval zebrafish, using an automated set-up. However, standardization of the set-up is essential given the fact that swimming behaviour of zebrafish larvae may be affected by both the intrinsic and extrinsic environment. Distance moved is the preferred parameter to express activity since velocity is, more than distance moved, hampered by several stimuli (intrinsic as well as extrinsic). This paradigm is suitable for rapid screening and can provide considerably more information on the behavioural effects of toxicants than other screening techniques; when applied in the context of REACH or early in the pharmaceutical drug development pipeline, this technique could ultimately lead to a reduction in animal use. Conflict of interest There is no conflict of interest. Acknowledgements Authors would like to thank Gerard van Beek and Dick Veldhuysen (Animal Facilities TNO Zeist) for taking care of the zebrafish. The constructive discussions (Didima de Groot) with Dr Ed Levin (Duke University Medical Center Durham, NC, USA) were very helpful to this research and much appreciated. The authors appreciate the comments of Dr. Jan Lammers, TNO Triskelion bv, Zeist, NL. This work was partly financed by The Netherlands' Ministry of Health, Welfare and Sports, and the Ministry of Social Affairs and Employment. In addition, part of the research was supported by The Netherlands' Ministry of Defence under R&T Program V936 ‘Military Toxicology’. References Alderton WK, Kimber GM, Richards FR, Strang I, Redfern WS, Winter MJ, Hutchinson TH, Hammond TG, Valentin JP. Validation of an optomotor method for assessment of visual function in zebrafish larvae. J Pharmacol Toxicol Methods 2008;58:169.

Arenzana FJ, Carvan III MJ, Aijon J, Sanchez-Gonzalez R, Arevalo R, Porteros A. Teratogenic effects of ethanol exposure on zebrafish visual system development. Neurotoxicol Teratol 2006;28:342–8. Barman RP. A taxonomic revision of the Indo-Burmese species of Danio rerio. Rec Zool Surv India Occas Pap 1991;137:1-91. Bhat A. Diversity and composition of freshwater fishes in river systems of Central Western Ghats, India. Environ Biol Fishes 2003;68:25–38. Bilotta J, Saszik S, Givin CM, Hardesty HR, Sutherland SE. Effects of embryonic exposure to ethanol on zebrafish visual function. Neurotoxicol Teratol 2002;24:759–66. Bilotta J, Barnett JA, Hancock L, Saszik S. Ethanol exposure alters zebrafish development: a novel model of fetal alcohol syndrome. Neurotoxicol Teratol 2004;26:737–43. Blader P, Strähle U. Ethanol impairs migration of the prechordal plate in the zebrafish embryo. Dev Biol 1998;201:185–201. Burgess HA, Granato M. Modulation of locomotor activity in larval zebrafish during light adaptation. J Exp Biol 2007;210:2526–39. Camarini R, Marcourakis T, Teodorov E, Yonamine M, Calil HM. Ethanol-induced sensitization depends preferentially on D1 rather than D2 dopamine receptors. Pharmacol Biochem Behav 2011;98:173–80. Carvan III MJ, Loucks E, Weber DN, Williams FE. Ethanol effects on the developing zebrafish: neurobehavior and skeletal morphogenesis. Neurotoxicol Teratol 2004;26: 757–68. Chen JN, Haffter P, Odenthal J, Vogelsang E, Brand M, van Eeden FJ, Furutani-Seiki M, Granato M, Hammerschmidt M, Heisenberg CP, Jiang YJ, Kane DA, Kelsh RN, Mullins MC, Nusslein-Volhard C. Mutations affecting the cardiovascular system and other internal organs in zebrafish. Development 1996;123:293–302. Colwill RM, Creton R. Locomotor behaviors in zebrafish (Danio rerio) larvae. Behav Processes 2011;86:222–9. Driever W, Solnica-Krezel L, Schier AF, Neuhauss SC, Malicki J, Stemple DL, Stainier DY, Zwartkruis F, Abdelilah S, Rangini Z, Belak J, Boggs C. A genetic screen for mutations affecting embryogenesis in zebrafish. Development 1996;123:37–46. Emran F, Rihel J, Adolph AR, Wong KY, Kraves S, Dowling JE. OFF ganglion cells cannot drive the optokinetic reflex in zebrafish. Proc Natl Acad Sci U S A 2007;104:19126–31. Emran F, Rihel J, Dowling JE. A behavioral assay to measure responsiveness of zebrafish to changes in light intensities. J Vis Exp 2008;20:923. Fei XC, Song C, Gao HW. Transmembrane transports of acrylamide and bisphenol A and effects on development of zebrafish (Danio rerio). J Hazard Mater 2010;184:81–8. Fischer KF, Lukasiewicz PD, Wong ROL. Age-dependent and cell class-specific modulation of retinal ganglion cell bursting activity by GABA. J Neurosci 1998;18:3767. Fraysse B, Mons R, Garric J. Development of a zebrafish 4-day embryo-larval bioassay to assess toxicity of chemicals. Ecotoxicol Environ Saf 2006;63:253–67. Gerlai R. Zebra fish: an uncharted behavior genetic model. Behav Genet 2003;33:461–8. Golling G, Amsterdam A, Sun Z, Antonelli M, Maldonado E, Chen W, Burgess S, Haldi M, Artzt K, Farrington S, Lin SY, Nissen RM, Hopkins N. Insertional mutagenesis in zebrafish rapidly identifies genes essential for early vertebrate development. Nat Genet 2002;31:135–40. Grunwald DJ, Eisen JS. Headwaters of the zebrafish – emergence of a new model vertebrate. Nat Rev Genet 2002;3:717–24. Helfman G. Fish behaviour by day, night and twilight. In: Pitcher T, editor. The behaviour of Teleost Fishes. Baltimore: John Hopkins University Press; 1986. p. 366–87. Hen Chow ES, Cheng SH. Cadmium affects muscle type development and axon growth in zebrafish embryonic somitogenesis. Toxicol Sci 2003;73:149–59. Hill A, Howard CV, Strahle U, Cossins A. Neurodevelopmental defects in zebrafish (Danio rerio) at environmentally relevant dioxin (TCDD) concentrations. Toxicol Sci 2003;76:392–9.

C. de Esch et al. / Neurotoxicology and Teratology 34 (2012) 425–433 Hurd MW, Debruyne J, Straume M, Cahill GM. Circadian rhythms of locomotor activity in zebrafish. Physiol Behav 1998;65:465–72. Irons TD, MacPhail RC, Hunter DL, Padilla S. Acute neuroactive drug exposures alter locomotor activity in larval zebrafish. Neurotoxicol Teratol 2009;32:84–90. Jerlhag E, Landgren S, Egecioglu E, Dickson SL, Engel JA. The alcohol-induced locomotor stimulation and accumbal dopamine release is suppressed in ghrelin knockout mice. Alcohol 2011;45:341–7. Kari G, Rodeck U, Dicker AP. Zebrafish: An emerging model system for human disease and drug discovery. Clin Pharmacol Ther 2007;82:70–80. Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF. Stages of EmbryonicDevelopment of the Zebrafish. Dev Dyn 1995;203:253–310. Knapik EW. ENU mutagenesis in zebrafish–from genes to complex diseases. Mamm Genome 2000;11:511–9. Laale HW. Culture and preliminary observations of follicular isolates from adult zebra fish, Brachydanio rerio. Can J Zool 1977;55:304–9. Levin ED, Chrysanthis E, Yacisin K, Linney E. Chlorpyrifos exposure of developing zebrafish: effects on survival and long-term effects on response latency and spatial discrimination. Neurotoxicol Teratol 2003;25:51–7. Lewis KE, Eisen JS. From cells to circuits: development of the zebrafish spinal cord. Prog Neurobiol 2003;69:419–49. Lewis MJ, June HL. Neurobehavioral studies of ethanol reward and activation. Alcohol 1990;7:213–9. Lockwood B, Bjerke S, Kobayashi K, Guo S. Acute effects of alcohol on larval zebrafish: a genetic system for large-scale screening. Pharmacol Biochem Behav 2004;77:647–54. Loucks E, Carvan III MJ. Strain-dependent effects of developmental ethanol exposure in zebrafish. Neurotoxicol Teratol 2004;26:745–55. MacPhail RC, Brooks J, Hunter DL, Padnos B, Irons TD, Padilla S. Locomotion in larval zebrafish: Influence of time of day, lighting and ethanol. Neurotoxicology 2009;30:52–8. Masur J, dos Santos HM. Response variability of ethanol-induced locomotor activation in mice. Psychopharmacology (Berl) 1988;96:547–50. Masur J, Oliveira de Souza ML, Zwicker AP. The excitatory effect of ethanol: absence in rats, no tolerance and increased sensitivity in mice. Pharmacol Biochem Behav 1986;24:1225–8. McGrath P, Li CQ. Zebrafish: a predictive model for assessing drug-induced toxicity. Drug Discov Today 2008;13:394–401. Miklosi A, Andrew RJ. The zebrafish as a model for behavioral studies. Zebrafish 2006;3:227–34. Milan DJ, Peterson TA, Ruskin JN, Peterson RT, MacRae CA. Drugs that induce repolarization abnormalities cause bradycardia in zebrafish. Circulation 2003;107:1355–8. Moens CB, Prince VE. Constructing the hindbrain: insights from the zebrafish. Dev Dyn 2002;224:1-17. Nasevicius A, Ekker SC. Effective targeted gene 'knockdown' in zebrafish. Nat Genet 2000;26:216–20.

433

Padilla S, Hunter DL, Padnos B, Frady S, MacPhail RC. Assessing locomotor activity in larval zebrafish: Influence of extrinsic and intrinsic variables. Neurotoxicol Teratol 2011;33:624–30. Parng C. In vivo zebrafish assays for toxicity testing. Curr Opin Drug Discov Devel 2005;8:100–6. Plaut I. Effects of fin size on swimming performance, swimming behaviour and routine activity of zebrafish Danio rerio. J Exp Biol 2000;203:813–20. Prober DA, Rihel J, Onah AA, Sung RJ, Schier AF. Hypocretin/orexin overexpression induces an insomnia-like phenotype in zebrafish. J Neurosci 2006;26:13400–10. Reimers MJ, Flockton AR, Tanguay RL. Ethanol-and acetaldehyde-mediated developmental toxicity in zebrafish. Neurotoxicol Teratol 2004;26:769–81. Samson JC, Goodridge R, Olobatuyi F, Weis JS. Delayed effects of embryonic exposure of zebrafish (Danio rerio) to methylmercury (MeHg). Aquat Toxicol 2001;51:369–76. Selderslaghs IWT, Hooyberghs J, De Coen W, Witters HE. Locomotor activity in zebrafish embryos: A new method to assess developmental neurotoxicity. Neurotoxicol Teratol 2010;32:460–71. Summerton J, Weller D. Morpholino antisense oligomers: design, preparation, and properties. Antisense Nucleic Acid Drug Dev 1997;7:187–95. Sylvain NJ, Brewster DL, Ali DW. Zebrafish embryos exposed to alcohol undergo abnormal development of motor neurons and muscle fibers. Neurotoxicol Teratol 2010;32:472–80. Tabakoff B, Kiianmaa K. Does tolerance develop to the activating, as well as the depressant, effects of ethanol? Pharmacol Biochem Behav 1982;17:1073–6. Talwar PK, Jhingran AG. Inland fishes of India and Adjacent Countries. Calcutta: Oxford & I.B.H. Publishing; 1991. Thompson RF, Spencer WA. Habituation: a model phenomenon for the study of neuronal substrates of behavior. Psychol Rev 1966;73:16–43. Tierney KB. Behavioural assessments of neurotoxic effects and neurodegeneration in zebrafish. Biochim Biophys Acta (BBA)-Mol Basis Dis 2010;1812:381–9. Tillitt DE, Papoulias DM. 2,3,7,8-Tetrachlorodibenzo-p-dioxin toxicity in the zebrafish embryo: local circulation failure in the dorsal midbrain is associated with increased apoptosis. Toxicol Sci 2002;69:1–2. Ton C, Lin YX, Willett C. Zebrafish as a model for developmental neurotoxicity testing. Birth Defects Res A Clin Mol Teratol 2006;76:553–67. Wilson SW, Brand M, Eisen JS. Patterning the zebrafish central nervous system. Results Probl Cell Differ 2002;40:181–215. Winter MJ, Redfern WS, Hayfield AJ, Owen SF, Valentin JP, Hutchinson TH. Validation of a larval zebrafish locomotor assay for assessing the seizure liability of early-stage development drugs. J Pharmacol Toxicol Methods 2008;57:176–87.