Behaviour 149 (2012) 10991123

brill.com/beh

Review
A comparison of the light/dark and
novel tank tests in zebrafish
Caio Maximino a,b, , Rancs Benzecry c , Karen Renata Matos Oliveira a ,
Evander de Jesus Oliveira Batista a , Anderson Manoel Herculano a,b ,
Denis Broock Rosemberg b,d,e , Diogo Losch de Oliveira b,d,e and Rachel Blaser f
a

Laboratrio de Neuroendocrinologia, Instituto de Cincias Biolgicas,

Universidade Federal do Par, Belm, PA, Brazil
b
Zebrafish Neuroscience Research Consortium, New Orleans, LA, USA
c
Faculdade de Biomedicina, Instituto de Cincias Biolgicas,
Universidade Federal do Par, Belm, PA, Brazil
d
Departamento de Bioqumica, Instituto de Cincias Bsicas e da Sade,
Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil
Instituto Nacional de Cincia e Tecnologia em Excitotoxicidade e Neuroproteo
(INCT-EN), Porto Alegre, RS, Brazil
f
Department of Psychology, University of San Diego, San Diego, CA, USA
* Corresponding authors e-mail address: caio.maximino@gmail.com
Accepted 15 October 2012

Abstract
The recent introduction of tasks to assess the behavior of zebrafish in novel and/or aversive environments has spurred great interest, prompting attempts to determine which constructs are modeled
by these tasks (e.g., fear, anxiety, or some other construct). A review of the pharmacological and
behavioral experiments indicates that not all behavioral testing models are equivalent. A more precise understanding of the parameters that influence task performance affords a wider selection of
experimental procedures for investigating a particular construct, and also provides tools for differentiating the various constructs that may ultimately be of interest. In this review we will more
closely examine two behavioral assays commonly used to measure the construct of anxiety in
adult zebrafish, with the conclusion that they do not both appear to be measuring a single underlying state.

Keywords
zebrafish, anxiety, fear, scototaxis, geotaxis.

2012 Koninklijke Brill NV, Leiden

DOI:10.1163/1568539X-00003029

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Scototaxis and geotaxis in zebrafish

1. Fear/anxiety
In the USA alone, anxiety disorders affect approximately 40 million adults
and cost more than $42 billion per year (Horwarth et al., 2011). Animal
models may be useful to understand how genetic and environmental factors
contribute to both normal and pathological anxiety, as well as which behavioral or pharmacological interventions may be effective treatments (Crawley,
1989; Green & Hodges, 1991; Rodgers et al., 1997; Gmez et al., 2002;
Kalueff & Tuohimaa, 2004; Kalueff et al., 2007). Moreover, research examining anxiety-related traits in humans may be a valuable resource for
understanding the evolution of defensive behavior in all animals (Rodgers et
al., 1997; Maximino et al., 2010a). In this context, the zebrafish (Danio rerio
Hamilton, 1822) is emerging as a potentially useful animal model of anxiety
and defensive behavior (Cachat et al., 2010; Gerlai, 2010, 2011; Maximino
et al., 2010b; Stewart et al., 2011b).
In humans, a distinction is typically made between state anxiety, which
is a measure of acute or immediate anxiety, and trait anxiety, which reflects
the long-term tendency of an individual to express anxiety in response to environmental events (Cattell, 1966). Anxiety disorders, including specific or
social phobias, generalized anxiety disorder, panic disorder, post-traumatic
stress disorder and obsessive-compulsive disorder, occur when normal fear
or anxiety responses become exacerbated and maladaptive. Because of the
commonalities between normal defensive behavior and pathological anxiety,
a comprehensive understanding of the genetic and developmental trajectories
of fear and anxiety especially trait anxiety may help to guide prevention and treatment of the disorders (Gould & Gottesman, 2006; Kalueff et al.,
2007). Animal models have proven to be extremely useful for understanding
the biological underpinnings of fear and anxiety, due in part to the practical and ethical difficulties of experimentation with human subjects, and in
part to the reduced complexity of animal models (Green & Hodges, 1991).
Conversely, because both normal and pathological anxiety are well-studied
in humans, information from clinical research may also be useful for a more
general understanding of the evolution of defensive behavior in animals.
In mammalian models, anxiety is typically differentiated from fear on the
basis of stimulus proximity and certainty. That is, fear is the response to immediate, tangible threat, while anxiety is a response to potential or distant
threat. In determining the construct(s) being measured in a novel test, therefore, it is necessary to understand which stimuli are controlling the defensive

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behaviors in order to manipulate proximity, probability, and degree of threat

or aversiveness (Maximino et al., 2010a). While the behavior of rodents in
response to both fear and anxiety has been extensively documented, research
with zebrafish is limited, and it is not yet clear which stimuli are best suited
to induce fear vs. anxiety, whether the two states produce different characteristic patterns of behavior, or whether a useful distinction can even be made
between the two.
Gray (1987) proposed five categories of fear-inducing stimuli for animals. The first comprises stimuli for which evolution has shaped a response
(i.e., predator stimuli). The use of predatory stimuli to induce fear in zebrafish has so far met with limited success (Bass & Gerlai, 2008; Speedie
& Gerlai, 2008; Gerlai et al., 2009; McHenry et al., 2009; Parra et al.,
2009; Ahmed et al., 2011; Jrgensen et al., 2012), although these models are
continually being improved (e.g., Luca & Gerlai, 2012a, b). The second is
novelty, in which an animal is confronted with an unfamiliar object or environment. When placed in a novel tank, zebrafish initially exhibit pronounced
behavioral inhibition (remaining near the bottom of the tank with limited
locomotor/exploratory activity), which gradually habituates with prolonged
exposure (Levin et al., 2007; Cachat et al., 2010, 2011a; Wong et al., 2010;
Rosemberg et al., 2011). The third category comprises classically conditioned stimuli that have been paired with aversive unconditioned stimuli,
such as a mild electric shock. Although, to date, modest research has been
conducted on classical and operant conditioning in zebrafish, animals learn
how to avoid a neutral stimulus that has been paired with a shock (Xu et al.,
2007; de Castro et al., 2008; Blank et al., 2009; Ng et al., 2012) which suggests that conditioned fear may be an effective approach. The fourth category
is fear produced by an intense stimulus, such as a sudden loud noise or bright
light. Zebrafish exhibit a characteristic and stereotyped startle response to vibrational or auditory cues (Kimmel et al., 1974; Eaton et al., 1977a, b; Hatta
& Korn, 1998; Bang et al., 2002; Burgress & Granato, 2007), although the interplay between startle and fear is not well studied. It may be that behavior in
the light/dark tank also falls into this category, since there is evidence that the
light side of the tank exerts aversive stimulus control which is modulated
by its brightness (Blaser et al., 2010; Maximino et al., 2010c; Blaser & Penalosa, 2011; Stephenson et al., 2011). The final category includes cues from
conspecifics. Alarm pheromones have been successfully used to elicit behavioral responses in zebrafish (Speedie & Gerlai, 2008; Egan et al., 2009; Parra

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et al., 2009; Mourabit et al., 2010; Wong et al., 2010; Gaikwad et al., 2011).
Social isolation induces a stressful response in zebrafish, which reflects in
fear/anxiety-related behavior in novel environments (Parker et al., 2012), and
research on shoaling behavior in this social species may also be useful for
understanding the role of social cues on fear and anxiety-related parameters
(Barba-Escobedo & Gould, 2012). Grays 5 categories of fear-inducing stimuli may all be effective for modeling fear or anxiety in zebrafish, but none
have yet been rigorously studied.
As with humans, differentiating between states and traits in animal models may improve the validity of the models. Boissy (1995) distinguished
between fear and fearfulness in animals, in analogy to the distinction between state and trait anxiety in humans. Boissy argued that fearfulness could
be considered a temperamental trait, based on correlations in behavior of
individuals across situations, and comparison of genetic lines selected for
behavioral traits. Evidence suggests that different zebrafish strains may respond differently to novel environments (Wright et al., 2003; Moretz et al.,
2007; Egan et al., 2009; Wisenden et al., 2011; Barba-Escobedo & Gould,
2012), raising the possibility that zebrafish exhibit genetically-linked trait
differences in fearfulness. Nonetheless, before such strain differences can be
attributed with any confidence to trait fear or anxiety, parallel results on additional tests of fear (such as fear conditioning or startle) are required in order
to rule out alternative interpretations.
2. The neural basis of anxiety and fear
In addition to the differentiation of fear and anxiety in terms of stimulus
control, there is evidence showing that these constructs are dissociated pharmacologically (Blanchard et al., 1993; Gray & McNaughton, 2000) and
neuroanatomically (Maximino, 2012) in mammals. While the response to
immediate threat is moderated by a network of structures which include the
central extended amygdala, the medial hypothalamic defense system, the
mesopontine rostromedial tegmental nucleus, and the periaqueductal gray
area (i.e., the fight-flight-freeze system), responses to potential threat are
mediated by a behavioral inhibition system composed of the medial prefrontal cortex, frontotemporal amygdala, ventral hippocampus and lateral
habenula (Gray & McNaughton, 2000; Maximino, 2012).
In contrast to the literature described for mammalian systems, there are
few reports on fear and anxiety-like responses in zebrafish, in part because

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the homologies between mammalian and teleostean neural structures are not
thoroughly determined (Maximino et al., in press). Lau et al. (2011) demonstrated that fish that consistently avoid the bright side of a light/dark tank
present significant c-fos mRNA upregulation in the medial zone of the dorsal telencephalon (Dm) and in the dorsal nucleus of the ventral telencephalon
(Vd), which are homologs to the mammalian basolateral amygdala and striatum, respectively. Okamoto et al. (2012) described that the habenula may
act as the multimodal switching board for controlling emotional behaviors
and/or memory inexperience dependent manners. The use of transgenic zebrafish, in which the neural signal transmission from the lateral subnucleus
of the dorsal habenula to the dorsal IPN was selectively impaired, revealed
that animals displayed enhanced levels of freezing response to presentation
of the conditioned aversive stimulus. Currently, a major goal in using the zebrafish should be to increase our knowledge of how the brain generates and
also terminates emotion (Guo et al., 2012; Jesuthasan, 2012). This understanding could occur at several levels. At the circuit level, the identification
of all cells involved (neural and glia, from the sensory system to the motor output neurons and stress circuitry), as well as characterization of how
information is transmitted and stored in the network is imperative (Guo et
al., 2012). Despite the differences on brain anatomy between teleosts and
mammals, future molecular approaches and the search for brain structures
that may be analog to mammalian ones (perhaps using image-based studies
and electroneurophysiological data) could improve the knowledge of riskassessment behaviors and fear/anxiety circuitry in zebrafish.
3. Anxiety as temperament
Behaviors are complex traits in the sense that any behavior is composed
of many subordinate traits at lower levels of biological organization (e.g.,
hormone levels, receptor distribution, neurotransmitter release, gene expression) (Sih et al., 2004a, b; Rale et al., 2007). The evolutionary response
to selection on behavior, then, requires associated changes in morphology,
physiology, and biochemistry (Koolhaas et al., 1999; Korte et al., 2005), and
is constrained by complex patterns of trade-offs (Wagner, 1996; Wagner &
Altenberg, 1996; Sinervo & Calsbeek, 2003; Korte et al., 2005). In general,
however, natural and sexual selection tends to act more strongly at higher
levels of biological organization (Wagner, 1996; Wagner & Altenberg, 1996;

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Garland & Kelly, 2006). At a genotypic level, alleles with complementary

pleiotropic effects should be favored, facilitating the coordinated evolution
of the hierarchically lower components of the behavioral trait in question
(Wagner & Altenberg, 1996). Thus, phenotypic variation in one trait results
from both among-individual allelic variability at loci with additive and nonadditive genetic effects, and from environmental variation (Roff, 1997). This
correlation structure leads to suites of behavioral traits that co-vary across
contexts or situations (Sih et al., 2004a, b; Moretz et al., 2007; Rale et al.,
2007), termed behavioral syndromes.
Quantitative genetic models suggest that the variance of a trait (sensu
Wagner, 1996) within and between populations of a given species is influenced by a relatively large number of genes with small individual effects, as
well as several environmental effects (Via & Lande, 1985; Roff, 1997; Ebstein et al., 2003; Sih et al., 2004a, b). Multiple traits can be associated at the
phenotypic level, suggesting potential genetic or epigenetic links between
them (Ebstein et al., 2003; Anholt, 2004; Joshi, 2005).
In humans, the Behaviorally Inhibited syndrome is similar to, and a risk
factor for, subsequent anxiety disorders. However, they may not be identical,
and both are still being investigated (Turner et al., 1996; Kagan & Snidman,
1999; Prez-Edgar & Fox, 2005). The Behaviorally Inhibited syndrome
is present in about 15% of the population, and is determined by an avoidance response to unfamiliar social and non-social stimuli. This behavioral
syndrome may be linked to high chronic activation levels of the biological
Behavioral Inhibition System (BIS), which is also associated with anxiety
and depression in humans (Gray & McNaughton, 2000; Corr, 2004; McNaughton & Corr, 2004; Hundt et al., 2007). Individual zebrafish or mutant
strains that exhibit a hyper- or hypo-active BIS could provide valuable insight into the genes and neural pathways involved in defensive behaviors.
Additionally, identifying inhibited behavioral syndromes may help clarify
the relationship between the underlying biological systems and clusters of
behavioral outcomes.
Experimental data suggest that anxiety can be polygenic and epistatic in
humans (Cloninger et al., 1998; Schinka et al., 2004; Hovatta & Barlow,
2008; Pezawas et al., 2008; Gadow et al., 2009; Montag et al., 2010). For
example, a quantitative trait locus (QTL) for anxiety-related syndromes has
been reported at chromosome 8p (Cloninger et al., 1998; Dina et al., 2005).
In laboratory mice, multivariate analyses increases the yield of QTL, with

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loci for 23 anxiety-related phenotypes being identified on chromosomes 3,

9, 13 and 17 (Henderson et al., 2004; Turri et al., 2004). In another analysis
of F2 hybrids of two strains of mice (A/J and C57BL/6) that differ in thigmotaxis and light avoidance, linkages of light-dark preference to chromosome
10 was found, with suggestive QTLs at chromosomes 6, 15, 19 and X (Gershenfeld & Paul, 1997); in the open field, suggestive QTLs were observed in
chromosomes 6 and 14 (Gershenfeld et al., 1997). The apparent contradiction between both studies can be explained by the use of genetically close or
genetically distant inbred strains of mice.
Besides QTL mapping, factorial analyses of multiple behavioral measures
have also been proposed. Ramos and colleagues (1997) analyzed the behavior of six inbred strains of rats in the open-field, the elevated plus-maze,
the light/dark preference test, and the social interaction test, reporting that
three factors (approach/avoidance towards aversive stimuli, activity in novel
environments, and a third factor with defecation in the open-field and social interaction) explain 85.1% of the variance within and between strains
and tests. Nonetheless, some associations (or lack thereof) were unexpected;
defecation and social interaction loaded positively on a separate factor, and
risk assessment measures loaded on the activity factor.
Similar studies have been made using zebrafish, although in a different
context and behavioral paradigms. Comparing wild-caught and inbred (AB
strain) zebrafish, Wright et al. (2006) found QTL for boldness (willingness
to approach a novel object; note that might be more appropriately categorized
as exploration/avoidance) at chromosomes 9 and 16, and QTL for shoaling
tendency at chromosomes 15, 18 and 24. The fact that these dimensions do
not map to the same QTLs is also supported by analysis of behavioral correlations in three different inbred strains (TM1, SH and Nadia), which revealed
no significant association between predator inspection and shoaling tendency
(Moretz et al., 2007). Again, the genetic distance between these strains might
be responsible for this lack of association. Alternatively, as we will suggest,
the multiple tests used do not reflect a single underlying construct, but different aspects of anxiety or defensive behavior systems (Ramos & Mormde,
1998; Ramos, 2008). These results highlight the difficulty of undertaking
genetic analysis of a behavior before the behaviors themselves are fully understood.
In the behavioral syndrome literature, two proposed categories may relate
to fear or anxiety-related temperaments. The first is the shyness/boldness

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dimension, which involves response to familiar risky or dangerous stimuli (e.g., a predator) (Conrad et al., 2011). The second is the exploration/avoidance dimension, which involves response to unfamiliar situations
or stimuli (e.g., a novel tank) (Conrad et al., 2011). This dimension corresponds quite well with the behavioral inhibition temperament in humans,
which is also measured in response to novel but not dangerous or aversive
stimuli (Barr, 2012). Although there is some evidence that the two dimensions are separable (individuals with high scores on one are not necessarily
high on the other), the distinction is primarily conceptual and may not truly
represent separate underlying constructs. Therefore, it is not clear whether
anxiety refers to the exploration-avoidance or to the shyness-boldness dimensions, or to something different altogether (Maximino et al., 2010a).
A possible relation between these dimensions and other theoretical models
of temperament/personality, as well as with anxiety disorders, is found on
Figure 1.

Figure 1. Theoretical model of the relationships between Eysencks PEN (psychoticismextroversion-neuroticism) model (Eysenck, 1991), reinforcement sensitivity theory (Corr,
2004), Cloningers biosocial model (Cloninger, 1987), the exploration-avoidance and
shyness-boldness dimensions of animal temperament (Rale et al., 2007), Hawk-Dove game
theoretical models (Korte et al., 2005), and anxiety disorders (Toth & Zupan, 2007). Abbreviations: BAS, behavioral activation system; BIS, behavioral inhibition system; FFFS,
fight-flight-freeze system; GAD, generalized anxiety disorder; OCD, obsessive-compulsive
disorder; PD, panic disorder.

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It is also possible that an anxiety syndrome lies at a hierarchically superior level of organization and is affected by both dimensions to some
degree. In rodents, it has long been noted that novelty can induce both exploratory behavior (curiosity (Berlyne, 1960)) and behavioral inhibition
(Montgomery, 1955; Russell, 1973). This motivational conflict became the
basis for the creation of a number of behavioral tests for anxiety, which involve exploration of novel environments (e.g., rodent open-field, elevated
plus-maze, light/dark box) (Rodgers et al., 1997; Maximino et al., 2010a).
Nonetheless, the apparatuses used in these tests usually set less aversive
(e.g., closed arms of the plus-maze, dark portion of the light/dark box)
against more aversive (e.g., open arms of the plus-maze, light portion of
the light/dark box) portions and, therefore, novelty is not the sole dimension
controlling behavior in these tasks (Toth & Zupan, 2007; Maximino et al.,
2010a). It could be argued that some setups currently applied to assess anxiety in zebrafish produce measures which fall along the exploration-avoidance
axis (e.g., open tank/novel tank test), while others produce measures which
fall along the shyness-boldness axis (e.g., light/dark test). On the other hand,
it can also be argued that, if behavior in both tasks is multidimensional, it
would be more correct to assign different behavioral endpoints within a given
task to different temperament dimensions (see Section 5 below for a more
extensive discussion of both tasks). This is still a matter for empirical research.
4. Stimulus control and validity
In devising any new measure or model, its validity must be established before useful results can be acquired. In particular, the meaning of a behavior is
often taken for granted at first, with difficulties then emerging when pharmacological studies demonstrate conflicting results. The construct validity, or
evidence that the measured behaviors are actually produced by (in this case)
anxiety, is the most difficult to establish. One difficulty is that anxiety as a
trait must be distinguished from specific reactivity to stimuli, such as greater
or less sensitivity to light, alarm pheromone, social isolation, and also from
individual variation in general locomotor output or specific motor responses.
One way to support the construct validity of a model is to establish convergent validity, or evidence that multiple test of anxiety all produce converging
results; that is, an anxious phenotype produces similarly high measures of

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anxiety-associated behaviors in multiple tests using a variety of stimuli and

responses. Failure to find convergence across tests suggests that the tests are
affected by multiple constructs or traits rather than a single underlying construct (Ramos & Mormde, 1998; Ramos, 2008; Maximino et al., 2010a).
Additionally, examination of stimulus control (which aspects of the test are
actually controlling the behavior) may help us to rule out specific sensitivities and determine which stimuli are most effective at inducing behavioral
change (Maximino et al., 2010a).
5. Measures/evidence for anxious behavior in zebrafish
5.1. Novel tank diving test
The novel tank test is a widely used method to assess the consequences of
stressful manipulations in zebrafish. This test was explicitly introduced as
an assay for anxiety by Levin et al. (2007), who reported that adult zebrafish spend about 50% of a 5-min session in the bottom of a novel tank
(bottom-dwelling), and that nicotine decreases this preference; anxiolytic
(anxiety-reducing) drugs, such as buspirone, diazepam and (chronic) fluoxetine, also decrease it, as well as the panicolytic (panic-reducing) drug tranylcypromine (Bencan et al., 2009; Egan et al., 2009; Stewart et al., 2011b).
Other studies showed that bottom-dwelling habituates over time (Bencan et
al., 2009; Wong et al., 2010), an effect which was abolished when animals
were tested in a tank with the same dimensions of the home tank (Bencan
et al., 2009). This habituation is also abolished by treatment with the anxiogenic drugs pentylenetetrazole and caffeine, as well as by pre-exposure to
the fear-inducing alarm substance (Schreckstoff) (Wong et al., 2010). This
latter manipulation also increased bottom-dwelling when animals are tested
individually (Egan et al., 2009), but not when animals were tested in groups
(Speedie & Gerlai, 2008, but see Jesuthasan & Mathuru, 2008) or when individual animals were exposed to hypoxanthine-3N-oxide (Parra et al., 2009),
a substance that is present in several fish alarm substances and that elicits
alarm reactions in other species (Pfeiffer et al., 1985; Brown et al., 2000).
An increase in bottom-dwelling response is also observed in zebrafish after
acute exposure to a cyanobacterium (Kist et al., 2011), acute restraint stress
(Piato et al., 2011b) and unpredictable chronic stress protocols (Piato et al.,
2011a). Both acute restraint stress and unpredictable chronic stress increase
bottom-dwelling as well as whole-body cortisol levels (Piato et al., 2011a, b);

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however, exposure to a cyanobacterium, which increases bottom-dwelling,

does not have the same endocrinological signature (Kist et al., 2011). Moreover, while there is a concordance between increased bottom-dwelling and
increased whole-body cortisol levels across pharmacological treatments, the
same is not necessarily true for anxiolytic manipulations especially using
psychotomimetic drugs (Grossman et al., 2010; Cachat et al., 2011a; Kyzar
et al., 2012).
The lack of reproducibility of the effects of an alarm substance or
hypoxanthine-3N -oxide could be due to low sensitivity of the test to feareliciting manipulations, or a characteristic of the type of manipulation induced. Indeed, exposure to a three-dimensional predator model (Blaser &
Gerlai, 2006), a computer-animated model (Gerlai et al., 2009) or to a live
predator (Bass & Gerlai, 2008; Cachat et al., 2011b) all fail to affect bottomdwelling, while presenting a computer-animated image of a bird silhouette
moving above the test tank strongly increases bottom-dwelling at the moment of stimulus presentation and after it (Luca & Gerlai, 2012a). This
suggests that bottom-dwelling may represent an important antipredatory behavior against aerial predators, which would be corroborated by evidence
that the diving behavior is motivated by avoidance of the top, but not approach to the bottom. Indeed, when animals are exposed to a tank with two
compartments differing in depth, a marked preference is observed for the
side that allows further escape from the surface (in one condition), while no
preference is observed for a side that allows closer proximity to the substrate
(in another condition; Blaser & Goldsteinholm, 2012). These results corroborate the idea that the motivation of the bottom-dwelling response does not
represent an approach to the bottom, but an escape from the waters surface.
Recent studies using distinct models of alcohol exposure have provided
evidence that bottom-dwelling in the novel tank test may reflect not a single construct (anxiety), but a combination of locomotor and motivational
effects. Ethanol exposure sometimes increases (Gerlai et al., 2008; Rosemberg et al., 2012), and sometimes decreases (Gerlai et al., 2008; Egan et al.,
2009; Mathur & Guo, 2011; Rosemberg et al., 2012), the diving response.
Recently, it was demonstrated that the duration of acute ethanol exposure
may be partially responsible for these actions, with 20 and 60 min administration inducing different effects on exploration, which were differently
antagonized by taurine pretreatments (Rosemberg et al., 2012). The combined results of these studies indicate that alcohol promotes two dissociable

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effects on behavior: one simply locomotor (stimulant-depressant/sedative)

and directly related to its brain concentrations; and the other motivational
(anxiolytic-anxiogenic) and unrelated to the brain ethanol content. These
results highlight the need for caution when interpreting a unidimensional
measure of behavior (duration in top or bottom of tank), and illustrate the
utility of a more integrative approach for analyzing the behavioral repertoire
of zebrafish.
5.2. Light/dark test
The light/dark test for zebrafish, in its present format, was initially proposed
by Serra et al. (1999) and further validated by Maximino and colleagues
(Maximino et al., 2007, 2010c) as a behavioral assay for evaluating anxietylike behaviors in small teleosts. Later pharmacological evidence suggested
that this assay is sensitive to anxiolytic, but not panicolytic, drugs (Maximino
et al., 2011). Although a preference for the darker portion of the apparatus has been widely replicated, behavior in this task is very sensitive to the
stimuli used. Gerlai et al. (2000) observed a preference for the light environment instead of a preference for darkness. This discrepancy could be likely
attributed to the different configurations of the tank used in this latter experiment, in which the white compartment was substituted for a transparent one,
and the dark compartment was created by covering the top as well as the
sides of the tank. Similar results were obtained by Champagne et al. (2010),
in which animals exposed to a black/transparent tank spent more time on
the transparent side, a preference which was abolished after restraint stress.
Similarly, animals spend more time near the transparent wall than the opaque
walls of an open-tank, and this preference is reduced after restraint (Champagne et al., 2010; but see Blaser & Pealosa, 2011). Other interesting results
were described by Steenbergen et al. (2011) for juvenile zebrafish, in which
they spent less time in the dark compartment relative to the white compartment of the testing apparatus. In the respective study, the authors verified that
dark-avoidance behaviors were significantly attenuated by commonly used
anxiolytic (diazepam, buspirone and ethanol) and increased by anxiogenic
drugs (such as caffeine), respectively. These results support the idea that the
anxious phenotypes in both juvenile and adult animals are distinct. Future
studies which aim to investigate the association between multiple behavioral
tests on different ages using a single protocol could provide interesting data

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about the mechanisms related to changes on defensive strategies during zebrafish lifespan.
Further emphasizing the role of the color and illumination on both sides
of the tank in establishing light/dark preference, Stephenson et al. (2011)
varied the light levels above the tank and showed that, at lower illumination
levels, animals spend more time in the black compartment, being indifferent
at intermediary light levels, and regaining preference for the black compartment at higher illumination levels. When increased light was concentrated
on the white compartment, dark preference increased (Arajo et al., 2012).
Similarly, animals preferred the dark portion of the apparatus when the other
portion is white, but not when the other portion is transparent with overhead
illumination (Blaser & Penalosa, 2011). Additionally, the expression of darkavoidance behaviors in juvenile zebrafish were reduced in a manner similar
to what is achieved with anxiolytic drugs, simply by decreasing the contrast
between the white and dark zones, which made the dark zone less dark. These
results suggest that manipulating the aversiveness of the light compartment
by changing light levels or compartment color leads to predictable changes
in the behavioral repertoire observed in this test.
In a similar vein, animals which show a high avoidance of the white compartment freeze more when confined in that portion than animals that present
low avoidance (Blaser et al., 2010). The confinement into a white compartment thrice before allowing the fish to freely explore the light/dark apparatus
does not change dark preference, but decreases erratic swimming, thigmotaxis and freezing behavior (Maximino et al., 2010c). Confinement into a
white tank (relative to confinement to black or transparent) prior to the novel
tank test decreases bottom-dwelling, the latency to enter the top of a novel
tank, and the time spent in a homebase (Rosemberg et al., 2011). In contrast, the diving response is reduced when tested during confinement to black,
relative to white or transparent (Rosemberg et al., 2011). Taken in combination, these results underline the aversiveness of the white compartment as an
important factor in controlling behavior in the light/dark test. Nonetheless,
they also suggest that escape from this compartment is not the sole motivation for this. Supporting this latter conclusion, it has been observed that white
avoidance does not habituate within or between sessions, while thigmotaxis
and freezing in the white compartment show a biphasic pattern (Maximino
et al., 2010c; Stewart et al., 2011a).

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6. Comparison and conclusions

The existence of an anxious trait in zebrafish would be most convincingly
demonstrated by evidence that individuals and genetic strains show correlations in anxiety-related behaviors (freezing, erratic movement, diving, shoaling) across repeated tests and different situations (novel tank, light/dark). So
far, no data is available to support or refute the existence of such correlations
and this issue is still unresolved. However, the combined results from the
novel tank and the light/dark test provide limited support for convergence
of the two tests, call into question whether they measure the same construct
(see Table 1 for a comparison of the two tests). Although both are sensitive
to anxiolytic/anxiogenic drugs, the pharmacological profiles are not always
identical. For example, the novel tank seems to be sensitive to diazepam,
but not to chlordiazepoxide, while the light/dark task is sensitive to other
benzodiazepines as well (Bencan et al., 2009; Maximino et al., 2011). Both
behavioral tests also show different results when animals are exposed to fluoxetine (Egan et al., 2009; Maximino et al., 2011), while the light/dark test
shows a lack of sensitivity for moclobemide, a MAO-A inhibitor (Maximino
et al., 2011). From a behavioral perspective as well, evidence is available
for a putative dissociation between the effects of color and depth stimuli on
zebrafish behavior (Blaser et al., 2010; Maximino et al., 2010c; Blaser &
Penalosa, 2011; Rosemberg et al., 2011; Stewart et al., 2011a; Arajo et al.,
2012; Blaser & Goldsteinholm, 2012; Blaser & Rosemberg, 2012). When a
specialized apparatus was used to examine interactions between color and
depth preference, it was found that manipulation of color stimuli produces
a different pattern of behavior than manipulation of depth stimuli (Blaser &
Rosemberg, 2012). The manipulations of both color and depth stimuli lead
to differential behavioral profiles, providing support that the effects of color
and depth stimuli on zebrafish behavior may not reflect a common mechanism.
Currently, the use of correlations and factor analyses to characterize multiple behavioral assays in zebrafish is underutilized. However, these approaches are absolutely critical to the future direction of this research. First,
the results of such studies can provide evidence for or against the validity
of these models, and, therefore, improve our understanding of what is being
measured in our tests. Second, by directly comparing across multiple tests
the effects of stress, drugs, genetic manipulations, or different populations

C. Maximino et al. / Behaviour 149 (2012) 10991123

1113

Table 1.
The effects of a variety of experimental treatments on behavior in the novel tank test and the
light/dark test.
Novel tank
test (time
in top)

Light/dark
test (time
in light)

References

Pharmacological experiments
Buspirone

Bencan et al. (2009); Gebauer et

al. (2011); Maximino et al.
(2011)

SSRIs
Fluoxetine (chronic)

Egan et al. (2009); Sackerman

et al. (2010); Wong et al.
(2010); Maximino et al. (2011)

ND

Citalopram
Morphine
Benzodiazepines
Clonazepam
Bromazepam
Diazepam
Chlordiazepoxide
Alcohol
0.25%
0.5%
1.0%
1.5%
MK-801
Caffeine

LSD

Nicotine

Other experiments
Predator and partial predator stimuli
Animated color photo (side)

Bird image or dot (above)

Bird image (side)

Alarm Pheromone

H3 NO (synthetic)

Stewart et al. (2010); Wong et

al. (2010)
Bencan et al. (2009); Gebauer et
al. (2011); Maximino et al.
(2011)

Gerlai et al. (2000, 2008); Egan

et al. (2009); Wong et al.
(2010); Mathur & Guo (2011);
Maximino et al. (2011);
Rosemberg et al. (2012)
Sison & Gerlai (2011)
Wong et al. (2010); Maximino
et al. (2011)
Grossman et al. (2010); Stewart
et al. (2010)
Levin et al. (2007); Sackerman
et al. (2010)
Hall & Subowski (1995); Korpi
& Wisenden (2001); Speedie &
Gerlai (2008); Egan et al.
(2009); Gerlai et al. (2009);
Wong et al. (2009); Ahmed et
al. (2011); Luca & Gerlai
(2011)

1114

Scototaxis and geotaxis in zebrafish

Table 1.
(Continued.)
Novel tank
test (time
in top)
Stressors
Unpredictable chronic stress
Confinement to
White/Transparent
Confinement to Shallow
Strain
AB
LD
LFWT
SFWT
Albino
Leopard
WIK

Light/dark
test (time
in light)

References

Blaser et al. (2010); Maximino

et al. (2010); Piato et al. (2011);
Blaser & Rosemberg (2012)

Gerlai et al. (2008); Egan et al.

(2009); Sackerman et al. (2010)

For simplicity, only the effects on duration (or proportion) in the upper portion of the novel
tank, and duration (or proportion) in the light side of the light/dark tank, are reported. In both,
a symbol indicates that the treatment increased duration in the top, or increased duration in
the light side, both indicative of anxiolytic effects. A symbol indicates that the treatment
decreased duration in the top or in the light side, indicative of an anxiogenic effect. A dash
indicates failure to find an effect, while blanks have not yet been tested. Multiple symbols
signify replicate studies. Also for simplicity, interactions (such as those between strain and
drug treatment) are not presented.

or genetic strains of zebrafish, evidence may be found for or against the existence of an anxiety trait or temperament in fish. Basic correlational data
of this nature may help to unravel the contributions of diverse genetic and
physiological factors at play in fear and anxiety. The evaluation of the effect
of several drugs, considering both time of exposure and concentration, associated to the increase of knowledge about their potential actions in teleost
CNS can be an exciting strategy for future approaches on the mechanistic
and neural basis of zebrafish behavior. Furthermore, the use of a behavioral
test battery is a potentially interesting direction for research, but developing
a single tank containing multiple types of stimuli may increase the reliability, efficiency (both in speed of experimentation and a reduced number
of animals), and comprehensiveness of the test for large-scale screening.
Therefore, the evaluation of neurochemical mechanisms involved in each be-

C. Maximino et al. / Behaviour 149 (2012) 10991123

1115

havioral task could provide additional evidence for construct and predictive
validities, the most important types of validity for making generalizations to
human behavior.
Acknowledgements
Part of the work reported in this paper was financed by CNPq/Brazil (grants
483336/2009-2 and 400039/2009-5). C.M. and D.B.R. are recipients of
CAPES studentships. A.M.H. is the recipient of a CNPq productivity grant.
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