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 TNO, Research group Quality and Safety, Zeist, The Netherlands b Erasmus MC, Department of Clinical Genetics, Rotterdam, The Netherlands abstract article info 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 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. Ef- fects 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 activ- ity 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 parame- ters 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 spe- cies in developmental biology is the zebraﬁsh (Danio rerio)(Barman, 1991; Bhat, 2003; Laale, 1977; Talwar and Jhingran, 1991). This fresh- water 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 organo- genesis 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 em- bryo allows easy developmental staging combined with functional 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 neuro- toxic 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 in- duced) toxicity screening purposes since they can live in small volumes, for example in a 384-wells plate, for a few days. Hydrophobic com- pounds 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 Neurotoxicology and Teratology 34 (2012) 425–433 ☆ 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: didima.degroot@tno.nl (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 Contents lists available at SciVerse ScienceDirect Neurotoxicology and Teratology journal homepage: www.elsevier.com/locate/neutera