Pharmacological study of the light/dark preference test in zebrafish (Danio rerio): Waterborne administration

Highlights

• We optimized the light/dark assay in zebrafish for anxiety evaluation.

• The light/dark assay was validated using a variety of drugs.

• Waterborne administration can be used to administer drugs in zebrafish.

• Zebrafish are a simple, cost-effective animal model to assess anxiety.

Abstract

Anxiety is a complex disorder; thus, its mechanisms remain unclear. Zebrafish (Danio rerio) are a promising pharmacological model for anxiety research. Light/dark preference test is a behaviorally validated measure of anxiety in zebrafish; however, it requires pharmacological validation. We sought to evaluate the sensitivity of the light/dark preference test in adult zebrafish by immersing them in drug solutions containing clonazepam, buspirone, imipramine, fluoxetine, paroxetine, haloperidol, risperidone, propranolol, or ethanol. The time spent in the dark environment, the latency time to first crossing, and the number of midline crossings were analyzed. Intermediate concentrations of clonazepam administered for 600 s decreased the time spent in the dark and increased locomotor activity. Buspirone reduced motor activity. Imipramine and fluoxetine increased time spent in the dark and the first latency, and decreased the number of alternations. Paroxetine did not alter the time in the dark; however, it increased the first latency time and decreased locomotor activity. Haloperidol decreased the time spent in the dark at low concentrations. Risperidone and propranolol did not change any parameters. Ethanol reduced the time spent in the dark and increased the number of crossings at intermediate concentrations. These results corroborate the previous work using intraperitoneal drug administration in zebrafish and rodents, suggesting that water drug delivery in zebrafish can effectively be used as an animal anxiety model.

Introduction

Anxiety is an emotion associated with risk assessment-like behavior toward potential threats through exposure to a new environment or a potential adverse stimulus in the environment (Graeff and Zangrossi Junior, 2010). In order to evaluate anxiety, behavioral tests must be sensitive enough to detect a wide variety of functional changes induced in the brain by psychological and pharmacological manipulations (Ahmed et al., 2011, Gerlai, 2010a). Thus, behavioral analysis is considered one of the best approaches to identify new pharmaceutical compounds for treating anxiety (Gerlai, 2010a).

Animal models play a crucial role in studying the mechanisms of anxiety (Ahmed et al., 2011, Cryan and Sweeney, 2011). A number of key characteristics are sought after in animal models, including (i) predictive validity that indicates that the animal model is sensitive to drugs with clinical efficacy; (ii) face validity, implying that the anxiety observed in the animal model is analogous to the behavioral and physiological responses observed in humans, and (iii) construct validity, relating to the similarity between the theoretical rationale underlying the behavior of the animal model and humans (Belzung and Griebel, 2001). The use of zebrafish (Danio rerio) as an animal model is increasing in several fields, including behavioral neuroscience (Ahmed et al., 2011, Blaser et al., 2010, Wong et al., 2010), toxicology, and pharmacology (Maximino et al., 2010a, Maximino et al., 2010b, Rico et al., 2011, Sumanas and Lin, 2004), because adult fish are usually small, inexpensive, and easy to maintain (Barros et al., 2008, Champagne et al., 2010, Gebauer et al., 2011, Gerlai, 2010b, Sumanas and Lin, 2004). Furthermore, zebrafish share several similarities with humans, including neurotransmitter content (cholinergic, 5-hydroxytryptaminergic, dopaminergic and noradrenergic) (Barros et al., 2008, Kim et al., 2004, Rico et al., 2011) and the presence of a nervous system containing a diencephalon, telencephalon, and cerebellum, and a peripheral nervous system with motor and sensory components, and enteric and autonomic nervous systems (Barros et al., 2008, Mathur and Guo, 2010). Further, zebrafish genome has been sequenced, and it shows genetic homology to that of humans (Barbazuk et al., 2000).

The light/dark preference test is based on the preference of adult zebrafish for dark environments (Serra et al., 1999). This behavioral analysis reflects the zebrafish's conflict between remaining in “safe” places – in this case, the dark environment, because the dorsal distribution of melanophores in the fish tends to minimize refraction and reflection of light, thereby reducing the visualization of predators – versus an innate motivation to explore novel environments. Although conflicted, the adult zebrafish shows a significant preference for dark compartment and avoids light (Mathur and Guo, 2010, Maximino et al., 2010b). The light/dark preference test has been validated in behavioral assessments (Maximino et al., 2010b) and has advantages over other tests (Maximino et al., 2011). However, the main disadvantage in relation to the open-field and the novel tank diving test is the lack of pharmacological validation (Maximino et al., 2010a).

Maximino et al. (2011) studied the effects of fluoxetine, clonazepam, diazepam, buspirone, ethanol, chlordiazepoxide, moclobemide, and caffeine on the light/dark preference in adult zebrafish after intraperitoneal administration. All drugs were administered acutely, with the exception of fluoxetine, which was administered acutely and chronically. The observed results of this experiment were similar to those obtained with rodent models using a light/dark box. However, intraperitoneal injection causes stress in animals (Maximino et al., 2010b, Stewart et al., 2011a). Thus, alternative methods were sought to identify anxiolytic drugs in zebrafish. Gebauer et al. (2011) showed that benzodiazepine, buspirone, propranolol, and ethanol increased the time spent in the light compartment in adult zebrafish when administered by immersion, which allows the molecules to be quickly absorbed via the gills or skin of the fish (Rihel and Schier, 2012). These data suggest that light/dark preference may be a practical, inexpensive, and sensitive screening method for anxiolytic drugs.

It is important to note that the test parameters used, including the tank (18 × 9 × 7 cm height × width × length) that is divided into two equal parts by a sliding door, the 3-cm water level, the initiation of experiments with the fish in the light compartment, and the 5 min test duration (Gebauer et al., 2011) differed from those validated by Maximino et al. (2010b). Their study used an acrylic tank (15 × 10 × 45 cm) divided equally into a black and white side, with a 10-cm water column, and central sliding doors that were colored with the same color as the box side. These doors defined a central compartment measuring 15 × 10 × 5 cm, and the test was initiated from this compartment following a 5-min habituation period. The sliding doors were then removed, allowing the zebrafish to explore the apparatus freely for 15 min.

Few studies have pharmacologically evaluated behavioral models to determine the appropriate tests for anxiety (Ahmed et al., 2011, Gerlai, 2010a). Thus, the present study sought to describe the sensitivity of the light/dark preference test in zebrafish to key drugs that influence anxiety (clonazepam, buspirone, imipramine, fluoxetine, paroxetine, haloperidol, risperidone, propranolol, and ethanol) delivered by acute immersion. To our knowledge, no similar studies in zebrafish have been performed previously.