Zebrafish models to study drug abuse-related phenotypes
Adam Stewart*, Keith Wong*, Jonathan Cachat, Siddharth Gaikwad, Evan Kyzar,
Nadine Wu, Peter Hart, Valerie Piet, Eli Utterback, Marco Elegante,
David Tien, Allan V. Kalueff**
Department of Pharmacology and Neuroscience Program, Zebrafish Neuroscience Research
Consortium (ZNRC), Tulane University Medical School, 1430 Tulane Ave., New Orleans, LA
70112, USA
*Both authors contributed equally
**Corresponding Author: Allan V. Kalueff, PhD
Department of Pharmacology, Room SL-83, Tulane University Medical School,
1430 Tulane Ave., New Orleans, LA 70112, USA.
Tel.: +1 504 988 3354. Email: avkalueff@gmail.com
1 Synopsis
Mounting evidence implicates the zebrafish (Danio rerio) as a promising model species for
reward and addiction research. Modeling drug abuse-related behavior in both adult and larval
zebrafish produced a wealth of clinically translatable data, also demonstrating their sensitivity to
various drugs of abuse and the ability to develop tolerance. Several studies have also applied
withdrawal paradigms to model the adverse effects of drug abuse in zebrafish. Here we summarize
recent findings of a wide spectrum of zebrafish drug abuse-related behavioral and physiological
phenotypes, discuss the existing challenges, and outline potential future directions of research in this
field.
Key words: zebrafish, drug abuse, withdrawal, tolerance, anxiety, stress, cortisol
2 Introduction
Drug abuse and addiction are serious mental health and societal problems [1-4]. They
represent complex brain disorders with multiple symptoms (Fig. 1) caused by both genetic and
environmental factors [5-9]. Various experimental (animal) models have been introduced to target
different aspects of drug abuse [10-14].
Zebrafish (Danio rerio) continue to emerge as a new promising model for reward and
addiction research [15-18]. Dopaminergic projections to zebrafish forebrain parallel the mesolimbic
system (implicated in drug addiction in mammals [19]), representing a pathway that is highly
conserved and develops at early ontogenesis [20]. Various behavioral paradigms have been
particularly useful in zebrafish addiction research (Table 1). For example, conditioned place
preference (CPP) and similar models reveal strong rewarding properties of different drugs in
zebrafish [18, 21]. Genetic factors also contribute to zebrafish behavioral responses [22],
demonstrating the link between individual genes and reward phenotypes (e.g., [18, 23]).
Modeling zebrafish behavior traditionally utilizes both adults and younger animals, including
larvae and older, free-feeding fry. While the distinction between larvae and fry is important, for the
purposes of this article, we will apply the term ‘larvae’ to both these stages. Overall, zebrafish larvae
display robust drug-evoked neurobehavioral phenotypes [24], and offer the ability to study multiple
animals simultaneously, within a high-throughput battery [24-26]. However, larval models do not
always display the complex behavior of their adult counterparts, and lack fully established mediatory
and endocrine systems [27], neural circuits [28] and neuromuscular systems [29]. Therefore, both
models should be used complementarily to study drug abuse-related neurobehavioral domains (Fig.
1).
3 A special attention must be paid to selecting the drug concentrations for zebrafish studies.
First, background literature is lacking for many psychotropic drugs, since the zebrafish is a new
model in behavioral pharmacology [30-32]. Second, although zebrafish possess all major
‘mammalian’ neurotransmitters, peptides and hormones [22, 33], the species differences in animal
physiology play a role. For example, unlike humans and rodents, zebrafish have have two forms of
serotonin transporter (SERT A and B) [34-36]. The blood-brain barrier of teleost fish is less
exclusive than in mammals, enabling serotonin to pass through it and affect both central and
peripheral mechanisms [37-39]. Thus, zebrafish may be differentially sensitive to various
serotonergic drugs, compared to other model species. Third, discrepancies are likely within different
zebrafish studies, since it is difficult to translate drug concentrations from larval into adult fish
models. Finally, because the method of drug treatment in most zebrafish studies is immersion
(instead of injection, as in other species), the effective drug concentrations may be based on different
pharmacokinetics than in rodents or humans. However, this does not necessarily represent a flaw of
a model, since water immersion may be advantageous for some drugs (e.g., that are quickly
metabolized or require prolonged treatment) and does not involve injection stress that may confound
behavioral data.
Sensitivity of zebrafish to drugs of abuse
Animal sensitivity to acute and chronic drugs of abuse (including both reward-related and
other behavioral effects) is an important phenotype (Fig. 1) [15-17, 40] which correlates with the
drug’s abuse potential. The sensitivity to drugs of abuse revealed strong genetic determinants of both
increased [45-47] and decreased [42-44] risks of drug abuse. Both animal and clinical models reveal
striking parallels in their sensitivity to cocaine [48], amphetamine [49], benzodiazepines [50],
ethanol [51], nicotine [52], opiates [53], and other psychotropic compounds [54, 55]. Zebrafish
4 models are also sensitive to a wide range of psychotropic compounds (Table 1). While these
responses will be only briefly discussed here, they generally parallel rodent and clinical
observations, thereby confirming the translational value of zebrafish models.
Larval zebrafish display an overt morphine preference, which is reduced by pre-treatment
with naloxone and abolished by blocking dopamine signaling with D1 antagonists [56]. Adult
zebrafish also show a strong dose-dependent preference for morphine [57], which strikingly
resembles rodent responses to this drug [58-60].
Zebrafish models have been extensively used to study the effects of ethanol. In larvae, acute
exposure to ethanol evokes hyperlocomotion at lower doses and a hypolocomotory effect at higher
doses [61]. Strain differences in larval ethanol responses have also been reported [61, 62]. A similar
U-shaped dose-response curve has been observed in adult zebrafish [16], also showing straindependent variations in their responses to ethanol [63], as well as reduced shoaling and increased
aggressiveness [64]. In contrast, chronic ethanol treatment has an anxiolytic effect on zebrafish
behavior [22] (Fig. 2A), also altering the expression of multiple brain genes, some of which are
implicated in the addiction [21] and similarly affected by ethanol in mammals [51, 65].
Nicotine exposure produces strong effects on zebrafish place preference and learning [21,
66]. Although learning and memory are not specifically addressed here, the established effects of
drugs of abuse that affect cognitive functions give further validity to the zebrafish model of drug
abuse [17, 40, 67, 68]. Chronic nicotine exposure in larval zebrafish leads to reduced swimming and
impairs their startle response [69]. In adult zebrafish, acute administration of nicotine has an
anxiolytic-like effect [70, 71] (also see Fig. 2B) similar to its effect in humans and rodents [52].
While zebrafish show a clear preference for cocaine in the CPP paradigm, there are also
several strains with a decreased sensitivity in this model [68, 72]. Overall, zebrafish CPP models
5 display a substantial similarity to rodent cocaine CPP studies [73]. Adult zebrafish treated acutely
with mild doses of cocaine display arousal (e.g., circling, fin extension), increased aggressiveness,
and decreased visual sensitivity [72]. However, higher concentrations of cocaine reduce fish
responses [72] in spite of the high brain cocaine levels [68].
The sensitivity of larval zebrafish to amphetamine [74] generally parallels a similar
locomotor response observed in mammals. While low concentrations of amphetamine increase
activity, higher concentrations of this drug markedly reduce zebrafish locomotion [17, 18]. The
rewarding properties of amphetamine have been reported in adult zebrafish in the CPP test [17], and
also parallel those seen in rodents [49].
Benzodiazepines are known to activate the reward system in rodents [50], and have also been
tested in zebrafish models. For example, both chlordiazepoxide and diazepam display anxiolytic-like
effects in adult zebrafish. While chlordiazepoxide increases exploratory behavior in the light/dark
box paradigm, it does not affect vertical localization in the novel tank test [75, 76]. In contrast,
diazepam increases exploration in the novel tank, exhibiting a biphasic response for low to moderate
doses [75].
Unlike their extensive use in rodent research, hallucinogenic drugs have only recently been
tested in zebrafish. For example, salvinorin A, one of the most potent hallucinogens, exhibits
rewarding properties in the CPP, accelerates zebrafish swimming in acute low doses, and reduces
locomotion (evoking low-velocity "trance-like" state) at high doses [77]. Recently resurrected
interest in psychedelic drug research [55, 78, 79] guided our group’s interest to testing these drugs in
zebrafish. Similar to other fish species’ responses to lysergic acid diethylamide (LSD) [80-83], adult
zebrafish produced significantly shorter latency to enter the top and fewer freezing bouts [67]. LSD
also caused significantly more top transitions and time spent in top, as well as elevated cortisol
6 levels, consistent with its well-known endocrine effects in humans [84] and animals [85, 86]. Acute
administration of another hallucinogen, 3,4-methylenedioxymethamphetamine (MDMA) produced
less clear-cut behaviors (Fig. 3B), albeit showing a similar reduction in freezing (data no shown).
While this may be relevant to known clinical effects of MDMA and LSD (including the
rewarding/euphoric behaviors [54, 55] and elevated corticoids [84-88]), further research is needed to
examine zebrafish sensitivity to various hallucinogenic drugs.
Zebrafish can also be used to study plant compounds with potential psychoactive properties.
For example, kratom (Mitragyna speciosa) is a medicinal leaf, often used as a tea for its calming and
energizing effects. Mitragynine, the major alkaloid identified from kratom, is a partial opioid agonist
producing similar effects to morphine in mammals [89]. When administered acutely, kratom extract
has a mild sedative effect in zebrafish novel tank (Fig. 4), but did not have psychoactive/anxiolytic
action over a wide range of concentrations (1-12 g/L) tested in our laboratory. This suggests that
zebrafish may display differential sensitivity to the drugs’ complex behavioral profiles.
Tolerance and withdrawal
Commonly observed for drugs of abuse in both clinical [90, 91] and animal [40, 92] studies,
tolerance is the progressive reduction of drug sensitivity, which requires higher doses to obtain the
same effects. Tolerance represents an important drug abuse-related phenotype (Fig. 1), mediated by
the brain’s adaptive mechanisms [93-95]. Recent studies have confirmed tolerance in adult zebrafish,
reporting that after chronic exposure to ethanol, zebrafish have a reduced response to the acute
effects of the drug [40]. Another study reported tolerance following chronic ethanol exposure, which
was also influenced by zebrafish genotype (strain) [96]. Tolerance is also seen in zebrafish following
chronic exposure to nicotine [21], collectively paralleling known rodent and clinical findings.
7 Withdrawal is another key phenotype associated with drug abuse [15] (Fig. 1), extensively
studied in various rodents following cessation of ethanol [97], cocaine [98], benzodiazepines [99],
and opiates [100]. These symptoms are also sensitive to various pharmacological [101, 102], genetic
[103] and behavioral [104, 105] factors. Withdrawal is believed to be mediated by the body’s
tendency to maintain homeostasis, in which counter-regulatory mechanisms produce unopposed
effects when a drug is abruptly removed [95, 106-110].
Researchers have recently turned their attention to studying the withdrawal phenomena in
zebrafish (Table 1). Acute discontinuation of drug treatment – acute (single) withdrawal – is a
common form of withdrawal, evoking strong behavioral effects in humans and rodents [111-115]. In
all studies, the most common behavioral manifestations of withdrawal include anxiety, seizures,
sedation, and pain [90, 116-119]. In zebrafish, acute ethanol discontinuation decreases zebrafish
shoaling behavior [120], whereas cocaine withdrawal evokes marked hyperlocomotion marked by
erratic movements and increased exploratory behavior [68, 121]. Acute discontinuation of ethanol,
diazepam, and morphine produces robust anxiogenic-like behavioral responses in zebrafish such as
hypolocomotion, decreased top transitions, and increased freezing bouts in the novel tank [15].
This novelty-based test [22, 71, 122] is an effective paradigm for observing withdrawal
behavior in zebrafish due to these clear-cut anxiety-like responses (see other papers in this issue for
details on zebrafish models of anxiety). Our experiments with chlordiazepoxide (CDP), a sedative
benzodiazepine known to produce anxiolytic effects in both rodents [123] and zebrafish [76], have
tested its withdrawal potential in zebrafish. Two groups of fish (n=15) were administered chronic
CDP (10 mg/L) for 4 months, with another groups of similar size used as a drug-free control. One of
the CDP-treated groups was withdrawn from CDP for 7 days before testing in the novel tank test
(Fig. 5). The withdrawal group displayed an increased latency to the top half of the tank, less
8 transitions to the top, a longer duration in the top, compared to controls. The withdrawal group also
exhibited an increased freezing duration, compared to both the control and the chronic CDP groups
(Fig. 5). Similar to the response observed in both humans [124-126] and rodents [127, 128],
withdrawal elevates zebrafish cortisol, correlating with the expected higher levels of stress and
anxiety [15] (also see Fig. 5 for CDP data).
In humans, chronic drug abuse represents a cyclical process of repeated reward and
withdrawal. Therefore, to more accurately model clinical withdrawal phenomena, repeated
withdrawal models are needed in addition to acute withdrawal studies. Repeated drug withdrawal
paradigms have been recently developed for rodents, showing that both the rat and human share
common triggers of relapse (such as the drug of abuse, stress, stimuli, or the environment
conditioned to the drug of abuse), and that withdrawal selectively potentiates responses to
anxiogenic stimuli [115, 129-132]. A recent study successfully applied repeated withdrawal protocol
to adult zebrafish, reporting that morphine and ethanol withdrawal leads to robust anxiety-like
behaviors [15]. Interestingly, not all drugs of abuse evoke withdrawal. For example, LSD (known for
its low ability to induce withdrawal in humans [133] and animals [134]) does not evoke withdrawal
in both acute and repeated zebrafish models (data not shown).
The complexity of withdrawal phenotypes [135-142], as well as the difficulty in modeling
withdrawal syndrome in animals [143-145], represent another challenge. Potentially interesting
directions of research may focus on neurochemical alterations, neural circuits, and the long-term
consequences [126, 146-148] of drug withdrawal in zebrafish. The genomic profiling of zebrafish
withdrawal also provides further insights, including altered gene expression in the zebrafish brain
following chronic drug treatment and withdrawal [21, 40, 120]. Sex differences in reported for
zebrafish withdrawal-related behaviors [121] parallel sex differences in human [149] and rodent
9 [117, 150-152] withdrawal responses, therefore increasing populational and construct validity of
these models.
Concluding remarks
Because drug abuse is a serious biomedical and societal problem, a better understanding of
the mechanisms behind drug reward and abuse is needed to develop innovate and more efficient
treatments. Considerable efforts have recently been made to develop reliable and high-throughput
assays for zebrafish behavior [17]. Mounting evidence indicates that zebrafish cognition is complex
and often parallel the abilities of mammals [122]. Since cognitive deficits commonly accompany
drug abuse (Fig. 1) [153-156], the ability to quantify both affective and cognitive phenomena in
zebrafish becomes important to study in relation to drug abuse phenotypes. Therefore, novel
behavioral models focusing on reward, cognitive and affective phenotypes may be needed to more
comprehensively model drug abuse in zebrafish.
We have previously emphasized the importance of promoting both larval and adult zebrafish
research [122]. The rich behavioral repertoire of the former complements the sensitivity and highthroughput nature of the latter [157]. Although the genetic aspects of addiction have long been
studied in mammalian organisms, zebrafish are also an ideal model for the use in forward genetics as
they can be rapidly cloned, have a relatively short generation time, and have large progeny sizes that
facilitate large-scale screens [17]. Further analysis of the molecular and biochemical pathways
underlying pre-existing genetic differences in zebrafish, such as strain-dependent variances, will
advance our understanding of how drug abuse affects the brain [62].
The ease of genetic manipulations and potential for high-throughput screening [21, 158, 159]
represent the strengths of zebrafish-based models. However, zebrafish models of drug abuse have
other important strengths, including behavioral complexity, 3-dimensionality, robustness of drug-
10 evoked phenotypes, and the ability to target a wide spectrum of neurobehavioral phenomena (Fig. 1,
Table 1). Therefore, a more comprehensive picture is needed in the field of zebrafish drug abuse
research, with a fair balance between their behavioral and genetic strengths. The growing availability
of new zebrafish strains through the Zebrafish International Repository Center [160], and current
progress of the Sanger Institute’s zebrafish genome project complements mounting support to
zebrafish behavioral research from the National Science Foundation, and recent cross-NIH programs
to develop new zebrafish genetic models and tools for their phenotyping. Together with multiple
lesser initiatives, such as international Zebrafish Neuroscience Research Consortium, this represents
a positive and promising development in the field.
Finally, we acknowledge the importance of applying cross-species and cross-domain
modeling [122] to drug abuse-related phenotypes (Fig. 1). For example, clinical drug abuse is highly
comorbid with anxiety [161], depression [162], bipolar disorder [163], and psychoses [164].
Therefore, animal models that simultaneously target these comorbidities, may lead to valid clinically
relevant experimental paradigms of drug abuse. Furthermore, since genetic variability influences
addiction sensitivity [62], the overlay between behavioral phenotypes and the propensity for, and the
expression of, addictive behavior must be considered. This approach may also improve the ability of
animal models to mimic the entire pathway of drug addiction, rather than a single point along the
continuum [165]. From this point of view, understanding the pathology of drug abuse and addiction
will benefit from implementation of innovative approaches and novel model species, such as
zebrafish.
Acknowledgement: This study is supported by Tulane University Intramural Research program,
Zebrafish Neuroscience Research Consortium (ZNRC) and LA Board of Regents’ P-Fund. The
11 authors thank J. DiLeo, C. Suciu, M. Hook, D. Carlos, K. Rhymes, K. Chang and L. Grossman for
their help with this study.
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A - adult zebrafish models; L - larval zebrafish models)
Cocaine
A
A
References
LSD*
Nicotine
A
Benzodiazepines
Ethanol
AL
Caffeine
Morphine
Domains
Amphetamine
Drugs
Reward
properties
[17, 21, 56, 57, 72, 166]
A
[16, 22, 56, 57, 61, 62,
Acute behavioral
effects
AL
AL
A
A
A
A
A
A
A
A
AL
AL
A
AL
A
A
67-69, 72, 74-76, 167]
Chronic
behavioral effects
A
[15, 22, 68]
A
Developing
tolerance
[15, 21, 40, 96]
A
Acute (single)
withdrawal
A
A
A
*
A
*
[15, 22, 68, 120]
Repeated
withdrawal
[15], Fig. 5
*Lysergic acid diethylamide (note that its acute or repeated withdrawal in our studies with adult
zebrafish failed to evoke overt anxiety, which is in line with clinically known weak withdrawalevoking potential of this drug).
21 Figure 1: Multiple inter-related domains of drug abuse (note that all these behavioral domains
are highly relevant to zebrafish models of drug abuse described here)
22 Figure 2. Examples of anxiolytic effects of chronic ethanol and acute nicotine in a 6-min novel
tank test. A - Chronic ethanol (0.3% for 1 week; n = 24 for each group). B - Acute nicotine
administration (10 mg/L for 5 min; n = 15 per each group). **P<0.01, ***P<0.001, U-test.
23 Figure 3. Examples of behavioral effects of hallucinogenic drugs in a 6-min novel tank test. A Acute lysergic acid diethylamide (LSD) administration (250 µg/L for 20 min; n = 10 per each
group). B - Acute 3,4-methylenedioxymethamphetamine (MDMA) administration (5 mg/L for
20 min; n = 10 per each group). ***P<0.001, U-test.
24 Figure 4. Examples of behavioral effects of kratom extract in a 6-min novel tank test. Fish
were acutely exposed to 6 g/L of kratom extract for 20 min (n=10 per each group).
25 Figure 5. Behavioral effects of 100 mg/L chronic chlordiazepoxide (CDP) in a 6-min novel tank
test. CDP was administered for 4 months, after which one group was withdrawn for 7 days (n
= 15 per each group). There was significant group effect for latency to upper half (F(2, 44) =
8.7, P<0.001), transitions to top (F(2, 44) = 5.2, P<0.01), time in top (F(2, 44) = 6.1, P<0.01) and
freezing duration (F(2, 44) = 14.7, P<0.001). *P<0.05, **P<0.01, ***P<0.001, #P=0.05-0.1
(trend) between the groups; post-hoc Tukey test for significant one-way ANOVA data.
26