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Adult zebrafish in CNS disease modeling: A tank that's half-full, not half-empty, and still filling

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The zebrafish (Danio rerio) is increasingly used in a broad array of biomedical studies, from cancer research to drug screening. Zebrafish also represent an emerging model organism for studying complex brain diseases. The number of zebrafish neuroscience studies is exponentially growing, significantly outpacing those conducted with rodents or other model organisms. Yet, there is still a substantial amount of resistance in adopting zebrafish as a first-choice model system. Studies of the repertoire of zebrafish neural and behavioral functions continue to reveal new opportunities for understanding the pathobiology of various CNS deficits. Although some of these models are well established in zebrafish, including models for anxiety, depression, and addiction, others are less recognized, for example, models of autism and obsessive-compulsive states. However, mounting data indicate that a wide spectrum of CNS diseases can be modeled in adult zebrafish. Here, we summarize recent findings using zebrafish CNS assays, discuss model limitations and the existing challenges, as well as outline future directions of research in this field. © 2017 Nature America, Inc., part of Springer Nature. All rights reserved.
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INTRODUCTION: ANIMAL MODELS OF CNS DISEASES
Neuropsychiatric diseases represent a difficult biomedical problem,
both in their investigation and treatment1,2. Traditionally, a limited
number of systems and processes has been targeted in CNS disease
modeling, including modulating brain circuits, neurotransmitter
systems3 and neuro-immune interactions4. However, many affected
CNS phenotypes (for example, social deficits in autism or cognitive
deficits in schizophrenia) lack efficient approved treatments5,6, and
the success rate for new drugs remains extremely low, despite the
rapidly growing body of relevant biological information7.
Preclinical research heavily depends on the use of animal
experimental models8–10. In vivo disease models are organisms
that express pathological phenomena observed in the actual human
disease, and can be used to understand how the disease develops
and, eventually, can be treated. Thus, animal models are crucial for
bridging preclinical and clinical research11. Usually, these experi-
mental models recapitulate only selected relevant aspects of dis-
ease, but not its full complexity, therefore markedly simplifying
the original disease2. The organisms used for such modeling may
show a different degree of similarity to the human disease of inter-
est, owing to either functional limitations of the animal, or poor
predictive power of current models. Furthermore, animal models
often cannot be used to evaluate the same endpoints as in human
clinical research, and therefore clinical feedback is becoming neces-
sary for preclinical research (Box 1).
CNS disease modeling also has the problem of delineating one
model from another, because the symptoms of the diseases and their
causes often overlap1,12–14. One alternative can involve modeling
a selected symptom or endophenotype that needs to be corrected,
rather than the ‘big’ disease syndrome per se. However, creation of
complex disease models, targeted to mimic multiple symptoms as
fully as possible, becomes critical for studying brain disorders2.
Another important alternative, recognized recently, is the use of
a wider spectrum of model organisms for CNS disease modeling.
Based on the premise of targeting evolutionarily conserved—and,
therefore, core—disease phenotypes and mechanisms, this strat-
egy highlights the value of novel model organisms, such as zebraf-
ish (Danio rerio), in translational neuroscience research15,16.
Indeed, zebrafish represent an emerging model organism for
studying complex brain diseases5,6,15,17–19, and a wide spectrum
of CNS diseases can be modeled in adult zebrafish, as will be dis-
cussed here using selected brain disorders as examples. While
some zebrafish models, including anxiety, depression, and addic-
tion-related models, are relatively well-established, others are
less recognized, such as models of autism and obsessive-compul-
sive states5,6,15,17. Additionally, although the number of zebrafish
1Institute of Translational Biomedicine, St. Petersburg State University, St. Petersburg, Russia. 2e International Zebrash Neuroscience Research Consortium (ZNRC),
Slidell, Louisiana, USA. 3Department of Behavioral Sciences, Arkansas Te ch University, Russellville, Arkansas, USA. 4School of Pharmaceutical Sciences, Southwest University,
Chongqing, China. 5Laboratory of Biological Psychiatry, ITBM, St. Petersburg State University, St. Petersburg, Russia. 6Ural Federal University, Ekaterinburg, Russia.
7ZENEREI Research Center, Slidell, Louisiana, USA. Correspondence should be addressed to A.V.K (avkalue@gmail.com).
Adult zebrafish in CNS disease modeling: a tank
that’s half-full, not half-empty, and still filling
Darya A Meshalkina1,2, Elana V Kysil1, Jason E Warnick2,3, Konstantin A Demin1,2 & Allan V Kalue 4–7
The zebrafish (Danio rerio) is increasingly used in a broad array of biomedical studies, from cancer
research to drug screening. Zebrafish also represent an emerging model organism for studying complex
brain diseases. The number of zebrafish neuroscience studies is exponentially growing, significantly
outpacing those conducted with rodents or other model organisms. Yet, there is still a substantial
amount of resistance in adopting zebrafish as a first-choice model system. Studies of the repertoire of
zebrafish neural and behavioral functions continue to reveal new opportunities for understanding the
pathobiology of various CNS deficits. Although some of these models are well established in zebrafish,
including models for anxiety, depression, and addiction, others are less recognized, for example, models
of autism and obsessive-compulsive states. However, mounting data indicate that a wide spectrum of
CNS diseases can be modeled in adult zebrafish. Here, we summarize recent findings using zebrafish
CNS assays, discuss model limitations and the existing challenges, as well as outline future directions of
research in this field.
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.
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LabAnimal Volume 46, No. 10 | OCTOBER 2017 379
neuroscience studies is exponentially growing, significantly outpac-
ing those conducted with rodents or other model organisms20, there
is still a substantial resistance in many laboratories in adopting zebraf-
ish as their first choice. Here, we summarize recent findings using the
zebrafish as an exciting novel tool to study complex CNS functions
and dysfunctions. We also critically discuss model limitations and
challenges (Ta bl e 1 and Tab le 2), as well as outline future directions
of research in this relatively young, but highly promising field.
The growing utility of zebrafish for biomedical research
Native to South-East Asia, the zebrafish is a small tropical fish21 inhab-
iting streams, canals, ponds and rice fields22. A popular aquarium
species, it has also been widely used in developmental, genetic23,24
and, now, neuroscience research25. Recent sequencing of the zebraf-
ish genome reveals high similarity to other vertebrates, with 71.4%
of human genes having at least one zebrafish ortholog26. However,
zebrafish underwent an additional round of teleost-specific whole-ge-
nome duplication and also contain multiple deletions, inversions and
duplications, compared to the human genome27. Clear advantages of
using zebrafish models (over rodents) include their low cost, relative
ease of their genetic manipulation6,16,28–30, and the capacity for non-in-
vasive drug administration, as water-soluble substances added directly
to water can be rapidly absorbed via gills and skin31,32. Zebrafish may
also be subjected to systemic drug administration, via intraperito-
neal33 or oral34 delivery, which can reduce the amount of drug used,
ensure better dosage control, and enable direct dose comparisons with
rodents. Additionally, introducing substances via immersion in fluid
helps avoid the injection stress that can often occur in rodents models.
Furthermore, approximate generation time of zebrafish is 3–4 months,
and females can spawn every 2-3 days producing ~200 eggs in each
clutch35. Zebrafish eggs are relatively large (0.7 mm in diameter), and
their larvae are optically transparent during the first days post ferti-
lization, enabling easy neonatal manipulations through all develop-
mental stages22. Fast reproduction, relatively high genetic homology
to humans, and low cost have made zebrafish a convenient and cost-ef-
fective tool for genetic, developmental and physiology research6,23,24,30,
as well as for high-throughput drug25,36 and toxicity screening37–39.
The increasing utility of zebrafish for CNS research
Zebrafish behavior is complex and relatively well-characterized40,
which becomes particularly useful for CNS disease modeling.
For example, zebrafish are highly social animals living in groups
(shoals) most of their lifetime41,42. Clearly, this trait is highly
relevant to disorders of human social behaviors, from aggression to
autism spectrum disorder (ASD)41. As diurnal species, like humans,
zebrafish are more reactive to visual stimuli than rodents43. Due
to the widespread use of zebrafish as pet and laboratory animals,
there are also various lines differing in skin or fin phenotypes (e.g.,
leopard, long-fin, short-fin and albino), as well as wild-caught
and established inbred and outbred laboratory strains (e.g., AB,
WIK, TM1 and TU), which differ markedly in their behaviors and
baseline levels of anxiety15,44. Such variability can promote stress
modeling and makes zebrafish a versatile and powerful model that
can be readily applied to study stress response mechanisms and
CNS drug discovery.
With the help of gene editing tools, a wide variety of zebrafish
knockins and knockouts have been created. Generating a zebrafish
line typically takes only one-third of the time needed for mouse
line creation45. A codon-optimized version of Cas9 endonuclease
for genome editing, recently developed for zebrafish, has proven
its elevated efficiency and specificity46. Full zebrafish embryo
transparency and high transparency of selected (e.g., casper) adult
zebrafish lines enables tracking of zebrafish development, tissue
discrimination and neural activity biomarkers, as well as optoge-
netic manipulation of this activity. Despite good conservation of
general vertebrate body and brain plan, brain anatomy is not fully
conserved between zebrafish and humans. For example, zebrafish
lack a prefrontal cortex and expanded telencephalon. However,
the functions that are attributed to these regions are present in
zebrafish, including complex cognitions16, mainly owing to the
development of subcortical neural circuits responsible for these
functions. Nevertheless, zebrafish possess all major neurotrans-
mitters, receptors and transporters, which are highly relevant for
targeting human CNS diseases and finding treatments15,16.
MAJOR CNS DISEASE MODELS IN ZEBRAFISH
Affective disorders
Affective disorders, including stress-related, anxiety spectrum,
depression, post-traumatic and phobic disorders are the leading
cause of human disabilities47, often accompanying other neurologi-
cal and psychiatric diseases. Zebrafish have recently been applied
to modeling affective disorders, and are rapidly gaining popular-
ity in this field16,48. This has fostered the development of multiple
zebrafish behavioral paradigms to study affective behaviors, most
of which resemble well-established rodent tests48,49.
The two most popular zebrafish behavioral paradigms, the novel
tank (NTT) and the light/dark (LDT) tests, represent conceptual
analogs of rodent novelty-based open field and light/dark tasks48,50,
which are all based on animals’ motivational conflict between innate
preference to stay in protective area and subsequently developing
exploratory behavior. During the NTT procedure, zebrafish are
typically placed into an unfamiliar narrow rectangular tank divided
virtually into two equal horizontal halves, and allowed to swim
freely for 5-30 min51,52. Fish initially spend more time in the bottom
Box 1 | Major challenges in CNS disease
modeling and treatment
Challenging, poorly understood, and complex neurobiology of higher
brain functions
Frequent comorbidity and shared/overlapping CNS pathogenesis
Unclear clinical symptoms of CNS diseases and problems with their
diagnostics
The historical tendency to treat brain and mind disorders separately,
as two branches (neurology and psychiatry)
Psychiatric diagnosis is based on behavioral symptoms that are
difficult or impossible to model in animals
Practical and ethical difficulties in examining human brain function
Lack of novel efficient neuroactive drugs targeting recently
recognized CNS pathways
Difficulties with classic high-throughput screening currently used for
CNS drug discovery
High failure rate of CNS drugs clinical trials (for example, see ref. 7)
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zone, show increased number of erratic movements or freezing
bouts (considered as anxiety-related behavior), but later, owing
to acclimatization, begin to gradually explore the top area of the
tank53. Behavior of model animals in such stressful conditions can
be analyzed manually by trained researchers or with specific soft-
ware. Automatized analysis significantly raises the throughput and
expands opportunities for registration of more endpoints in com-
parison with manual analysis28,36. In addition to the most widely
used elements of behavior which can be measured by the observer
(e.g., time spent in the top or bottom halves of the tank, number
of entries to the top area, latency to enter the top part, number
of erratic movements, duration of freezing, bouts) automatic
analysis can register swim velocity, turn angles, covered distance
and, moreover, make possible tracks’ of 3D reconstructions, even
with color mapping of velocity parameters in the manner of a
heatmap52,54–56. In comparison with the rodent open field para-
digm, zebrafish NTT also measures the vertical component of
exploratory behavior, resulting in a ‘true’ 3D analyses of affective
behavioral phenotypess28,36.
The LDT paradigm uses the natural tendency of wild zebrafish to
hide from predator aggression in the dark areas. Similar to rodents,
adult zebrafish tend to avoid lit areas and spend significantly more
time in darkness50. A typical apparatus for LDT is a tank consisting
of two vertical chambers identical in size, but differing in colora-
tion of walls: white and black (or transparent and black)49,52,57. The
principal idea of this paradigm is that the more anxious fish are,
the more time they will spend hiding in the dark part of the tank50.
Main endpoints used in this test are time spent in lit/dark areas,
the latency to enter the light area, shuttling (the number of total
transitions between the two compartments), and the number of
risk assessment episodes (e.g., fast entries to the lit area followed
by re-entry to the black compartment, as well as ‘partial entries’ to
the white compartment)58.
The open field test for zebrafish is a direct analog of the same
rodent paradigm, developed to investigate the horizontal compo-
nent of locomotion and exploratory behavior strategies59. An appa-
ratus for the aquatic open field test typically comprises shallow tanks
that may differ in size, shape and color. For instance, a rectangular
tank with the bottom divided into several square zones, a white or
transparent cylinder with divisions into the peripheral (near to the
wall) and central zones of the water column52,56,60. Main behavioral
endpoints in this test include the time spent in the periphery or
center of the tank, distance traveled in each zone in the tank, the
number of transitions between zones, velocity in each zone of the
tank, and the number of freezing bouts and time spent frozen52,60.
Other tests aimed to evaluate anxiety are not so widely used in
the affective disorders modeling, but can be useful additions to these
three popular paradigms. Generally, they can be divided into three
main groups: (1) tests related to social behavior, (2) tests assessing
affective behaviors in response to the predator, and (3) novelty-
based tests using unfamiliar objects presentations. Despite the fact
that paradigms from the first group (shoaling and social preference
tests) target alteration in zebrafish social behavior, they can be used
for studying affective disorders42,61–63 because increased anxiety
affects zebrafish responses to con- and heterospecifics presented
through transparent walls and increase shoal cohesion52,56.
Other paradigms, such as predator avoidance and predator expo-
sure tests, further help dissect anxiety-related behavior following an
exposure to a natural predator or its still/moving images and robotic
models19,25,64. Likewise, zebrafish boldness, novel object approach,
and food neophobia tests examine fish reaction to the presentation
of unfamiliar objects, which can increase anxiety40,65,66.
Besides physical and psychological stressors, such as a chronic
unpredictable stress battery67, which can include net chasing68,
beaker stress2, predator exposure25 and alarm substance69, zebrafish
behavior can be modulated with a diverse range of pharmaceutical
agents that influence anxiety-related behavior. Traditional anxio-
lytic substances, such as fluoxetine70,71, diazepam71, buspirone72,73,
nicotine51 and acute ethanol74, are widely used in zebrafish affective
neurobehavioral research. Commonly used anxiogenic manipula-
tions in zebrafish studies include caffeine75, pentylenetertrazole,
ethanol withdrawal76 and yohimbine77. Moreover, zebrafish anxi-
ety-assessing tests have become invaluable high-throughput tools
for recently developed CNS drugs with only predicted, but incom-
pletely understood, effects15.
In summary, zebrafish represent a promising model to study
affective disorders, such as anxiety and depression30,78–80, due to
robust behavioral phenotypes and high genetic and physiologi-
cal homology with human endocrine responses to stress81,82. In
experimental conditions, fish can be subjected to the variety of
stressors, including chasing, crowding, disturbance of the light:
Table 1 | Selected significant limitations of zebrafish models in
CNS research
Model limitations Description
Genome Some mammalian genes are represented in zebrafish
in two copies (genome duplication). Despite high
genetic homology to rodents and humans, signifi-
cant genetic differences may affect some studies
Strains It is difficult to create and maintain genetically
identical lines in zebrafish
Sex The zebrafish has no sex chromosomes (like
humans) but has polygenic sex determination
Physiology Despite high physiological homology to humans,
significant differences remain and may have an
impact on some studies
Circuitry The lack of understanding of how the zebrafish
brain generates various behaviors
Methodology The field is still poor standardized, and specific
details of experimental environments have to be
described with thoroughness to be reproducible.
Additionally, there is a need in developing more
tests and novel paradigms for zebrafish neurobehav-
ioral research, including both fish-specific and those
adapted from rodent models
Behavior While major behavioral domains in zebrafish are shared
and conserved with mammals, various key behavioral end-
points have to be characterized in detail and described
General Zebrafish studies are also affected by standard transla-
tional neuroscience limitations (see Box 1 for details)
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dark cycle, changes in water pH, exposure to bright light, restraint
stress, or social isolation79. Chronic application of stressors results
in zebrafish models of anxiety and depression67. Zebrafish also
show substantial similarity to humans in the immunological char-
acteristics of anxiety, with elevated expression of pro-inflammatory
markers such as COX-2 and interleukin 6 (IL-6)70.
Autism
Autism spectrum disorder (ASD) is a lifelong neurodevelopmental
illness with early-onset social, motor and cognitive deficits that
may be comorbid with intellectual disability, psychopathology and
epilepsy83. The severity of ASD varies from mild to severe, dramati-
cally interfering with quality of life83. The estimated prevalence of
ASD is around 1.0–2.6% globally, depending on the diagnostic cri-
teria84,85. ASD pathogenesis depends on both genetic determinants
(including more than 600 genes currently associated with autism86)
and multiple pre and postnatal environmental factors87.
Zebrafish innate preference for conspecifics provides a model
of ASD by enabling measurement of core social deficits via
assessment of the distance between the test fish and the image of
Table 2 | Common problems facing zebrafish neuroscientists
Common misconceptions and problems Important considerations
Zebrafish are just ‘smaller mice’. Zebrafish provide an important reference point with millions of years of evolutionary
distance from mammals.
Zebrafish research offers little novel insight. Zebrafish can help delineate shared, evolutionarily conserved, and therefore core dis-
ease pathways, circuits, and mechanisms.
Zebrafish are not similar enough to humans. Zebrafish share ~75% of genes with humans.
Zebrafish possess completely different physiology. Although there are of course important differences, there are also striking similari-
ties between most of the major physiological and organ systems, including all major
CNS neurotransmitters and cell types.
Only larval fish should be used in research. Most brain diseases are disorders of adults, and therefore adult fish may be more
feasible for CNS disease modeling. There is also growing evidence that using adult
zebrafish for CNS drug screening and behavioral research8,22.
Zebrafish are not suitable for complex disease modeling. They have excellent cognitive and affective phenotypes.
Zebrafish behavior is simple and instinctively driven, and is there-
fore not suitable for modeling complex behaviors.
Incorrect. They have a wide spectrum of complex spontaneous behaviors in almost
every domain recorded in mice and humans (see ref. 27 for a comprehensive review).
Zebrafish brain is very different from that of mammals. There is a striking functional and anatomical similarity in zebrafish and mammalian
brain anatomy and physiology.
Zebrafish do not feel pain, and are inappropriate for pain research. They have similar nociceptive mechanisms as rodents and humans.
Zebrafish have no emotionality. Zebrafish display a wide spectrum of affective behaviors (see refs. 27,42,69 for details).
Zebrafish have no cortex, and their use is therefore very limited in
CNS research.
While true, many complex behaviors (and their underlying circuits) in CNS diseases
do not require cortical regulation, and therefore can still be studied in zebrafish.
Zebrafish use is against the 3Rs principle. Owing to higher phenotypic sensitivity, the required sample sizes for zebrafish
research can be smaller than in rodent experiments Also, zebrafish are a less sentient
species, thereby fully meeting the 3Rs principles.
Zebrafish behavior is poorly characterized and understood. See ref. 27 for a comprehensive summary of all zebrafish behaviors.
Fish behaviors are not rodent or human behaviors. While of course this is true, one should therefore focus on a large number of common
behaviors, as well on those disordered phenotypes and syndromes that do not require
direct face homology of human, rodent and fish behaviors (e.g., do not tail-suspend
fish to assess their ‘despair’).
Zebrafish behavior is hard to record and analyze. In addition to easily-detected behaviors that can be recorded even manually, most
of zebrafish behaviors can now be efficiently analyzed using automated videotrack-
ing techniques (even using the same tools for fish and mice), including both
high-throughput multi-animal recording28,140 and complex 3D-based ‘automated’
extraction and dissection of phenotypic data23,44.
Fish are not approved by FDA for preclinical drug-screening. Although in some cases this is true, researchers should continue to push to make
drug-screening with zebrafish more common.
There are no (or few) other fellow zebrafish investigators around,
to support my research.
The number of zebrafish laboratories will continue to grow, as will the number of
investigators. Network cross-institutionally, nationally and internationally.
My grant is not funded ‘because of zebrafish focus’. Don’t give up, and resubmit. Volunteer to serve on study sections, editorial boards
and review panels, to represent the field. Educate your colleagues and advocate for
your model.
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conspecifics, especially because zebrafish react readily to such vis-
ual stimuli88–91. Another widespread paradigm examines shoaling
formation by assessing the average distance between the members
of a homogeneous92,93 or heterogeneous94 shoal and the direction
of the shoal movement40. Inhibitory avoidance, the test used to
assess emotional learning, is also applicable to ASD modeling,
where an unconditionally attractive chamber of the test tank is
associated with an aversive stimulus95, measuring the latency to
enter the previously preferred compartment. Likewise, zebrafish
aggression can be tested in a variety of settings96, measuring, for
example, chasing, biting, fin raise, mouth opening, head butting and
other endpoints40. One should also note the simplest variant of this
paradigm, the flat mirror test, applied to ASD modeling recently88.
Clearly, further research investigating features of zebrafish social-
ity may give rise to new types of zebrafish ASD models, including,
for instance, testing facilitation of learning in groups97, establish-
ing and recognizing the group hierarchy98, social eavesdropping99
and learning generalization100. With the establishment of such
tests for zebrafish ASD models, scientists will get a wider variety of
instruments to analyze and compare pathogenesis between human
and fish more easily. Similarly, perseverative behavior – another
core ASD phenotype – can be measured in zebrafish by advanced
3D ethogram analysis101,102, which can assess not only novelty-
induced anxiety (discussed above), but also ASD-related perse-
verations, such as circling103, stereotypic mouth opening, and
corner alternation40.
Pharmacologically, ASD can be modeled as a memory impair-
ment (glutamatergic antagonist MK801; see ref. 94) or dopamin-
ergic impairment (D1 receptor antagonist SCH23390; see ref. 104).
Developmental exposure to toxins have also been studied in
zebrafish as models of ASD, including anti-epileptic drugs (e.g.,
valproic acid88), pesticides (e.g., chlorpyrifos105) and pollutants (e.
g., water-soluble fraction of crude oil103). Finally, zebrafish have
emerged as an excellent model to study genetic causes of ASD.
For example, zebrafish have orthologs of roughly 67% of human
genes that are implicated in autism. The mutation of several of these
genes has been useful for ASD modeling in zebrafish. Specifically,
the fmr1 knockout is the most behaviorally well-characterized
zebrafish strain relevant to ASD, including open-field hyperac-
tivity and impaired emotional learning106. There is also a group
of genes implicated in ASD pathogenesis, but only teratologically
characterized in zebrafish embryos. For instance, kctd13 sup-
pression induces macrocephaly and excessive cell proliferation in
zebrafish107, whereas a knockdown of auts2 reduces their brain
size and neuron count108. Chromodomain helicase DNA-binding
protein 8 (Chd8) morphants have macrocephaly, ectopic sites of
neuron progenitor localization109 that is supported by Cas9-knock-
down data110. However, further translational neurobehavioral
research is needed to target these and other ASD-related genes
in zebrafish models, including both ‘human-to-fish’ and ‘fish-
to-human’ search strategies.
Addiction
Addiction is a complex disorder with strong genetic and environ-
mental determinants. Aspects of drug abuse include sensitization,
tolerance, withdrawal, drug seeking, extinction, and relapse, and
have been efficiently recapitulated in zebrafish76,111–114. The most
widely used test to assess addiction development in animals is con-
ditional place preference that assesses the rewarding properties of
drugs114,115, applicable for both adult and juvenile zebrafish116, in
the same manner as it is commonly used in rodents. For example,
zebrafish are sensitive to all major drugs of abuse, and these effects
are generally similar to those obtained in humans or rodents111.
Zebrafish form addictions and show withdrawal syndrome to
ethanol76,117, nicotine118, cocaine119, morphine120 and ampheta-
mine121. The effects of drug withdrawal are usually observed in
anxiety testing, such as NTT discussed above. The main effects of
withdrawal in zebrafish include anxiety, sedation, shoaling disrup-
tion122, seizures, and arousal76. The repeated withdrawal model
is more clinically relevant, albeit sometimes more challenging to
establish, than single withdrawal74,76,111,117. Interesting (and poten-
tially clinically relevant), sex differences in withdrawal symptoms
have also been reported in zebrafish, since, for example, male fish
develop more robust cocaine withdrawal phenotype, while females
develop withdrawal more rapidly113.
NOVEL EMERGING CNS DISEASE MODELS IN ZEBRAFISH
Attention deficit hyperactivity disorder (ADHD)
Attention deficit hyperactivity disorder (ADHD) is a widespread
neurodevelopmental disorder characterized by impulsive motor
behavior, deficit of sustained attention, and hyperactivity in a famil-
iar context123. The prevalence of ADHD is estimated as 5–7% in
children and 2.5–5% in adults124. ADHD patients often have lower
brain volume, especially in prefrontal cortex, basal ganglia, cor-
pus callosum and cerebellum125. The disorder is usually treated by
stimulants126, such as the dopamine transporter methylphenidate,
which shows efficiency in nearly 70% of cases127. Genetic studies
implicate multiple genes in ADHD, including genes for dopamine,
norepinephrine, serotonin receptors, and transporters (e.g., the
DRD4 and COMT genes,) as well as genes of neurodevelopmental
factors (e.g., CDH13 and DISC1 genes)13. In general, animal models
of ADHD must fulfill several validity criteria128. For example, they
should demonstrate high face validity by ensuring that impulsivity
gradually develops over time, sustained attention-deficit appears
when stimuli are widely spaced over time, and hyperactivity is
underrepresented in novel conditions128. High construct valid-
ity necessitates altered reinforcement of novel behavior and defi-
cient extinction of previously reinforced behavior, to better mimic
ADHD and the gradual pathogenesis that is seen clinically128.
The most recognized rodent model of ADHD is spontaneously
hypertensive rats (SHR)129 with high distractibility, motor and cog-
nitive impulsivity, and increased overall variability in behavior130.
Another widely used model is the dopamine transporter (DAT)
knockout mouse131, which is hyperactive in novel situations, and
has impaired learning and memory. Despite the lack of DAT, these
animals are sensitive to serotonin system stimulants used for ADHD
treatment131. Common toxicological models of ADHD include 6-
hydroxydopamine–lesioned rats, which display hyperactivity that
characteristically increases after habituation to novelty and is alle-
viated by an anti-ADHD drug methylphenidate132. In zebrafish,
lead exposure evokes neurological deficits, including lower syn-
aptic density and axon guidance protein expression, but without
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aberrant behavior reminiscent of ADHD133. Some mutations
of ADHD-linked genes were also introduced in zebrafish. For
example, the mutants for two tyrosine hydroxylase, generated dur-
ing a large-scale mutation screening project of Sanger Institute in
2013, have yet to be behaviorally phenotyped134. Zebrafish with
morpholino knockdown of the latrophilin 3 gene demonstrate lar-
val motor hyperactivity with bursts of rapid swimming135. This
phenotype was supported by the misplacement of dopaminergic
neurons in the ventral diencephalon, and rescued by clinically effi-
cient anti-ADHD drugs methylphenidate and atomoxetine treat-
ment, increasing the validity of latrophilin 3 knockdown for ADHD
modeling in zebrafish135.
Finally, since ADHD is the disorder of impulse control, this can
be evaluated in zebrafish using the aquatic 5-choice serial reaction
time task136. During this procedure, the fish choose the correct arm
of the maze and wait before the light goes on, in order to obtain a
food reward. Thus, the choice of correct arm reflects the learning
abilities of the fish, and the waiting assesses zebrafish impulsivity
control abilities136. The test’s sensitivity to the clinically used anti-
ADHD drug atomoxetine (which predictably decreases the rate of
anticipatory responses in zebrafish) further supports the validity
of this model136.
Obsessive-compulsive disorder (OCD)
Obsessive-compulsive disorder (OCD) is a serious, debilitating
neurobehavioral disorder with polygenic nature and a 3-% lifetime
prevalence14. Symptoms of OCD are distressing, and include obses-
sions (unwanted thoughts, images, impulses or fears) and compul-
sions (repetitive, ritualized behaviors or mental acts) that trigger
each other83. OCD is categorized into two main subtypes: early-
onset and tic-related, which differ in both clinical outcome and
genetic background. The core mechanism of OCD is the dysfunction
of the brains motor inhibitory control system, which involves pre-
frontal cortex, striatum, globus pallidum and thalamus137, and
which can cause delayed response inhibition on a response inhibi-
tion task. The two main approaches to OCD treatment (cognitive-
behavioral therapy and serotoninergic antidepressants) have limited
efficiency, and OCD often remains chronic, treatment-resistant and
even aggravated. Genetic causes of OCD include genes relevant to
neurotransmitter systems (e.g. 5-HTTLPR, HTR2A, COMT and
MAOA), immune reactions and white matter maintenance14, with
each gene making small, incremental contributions that increase
the possibility of the disease.
Usually, the available animal models of OCD, which are typically
rodents, target stereotypic behaviors, such as repetitive, excessive,
topographically invariant movements that lack any obvious function
or purpose (e.g., grooming or chewing)10,137–139. The drawback of
such models is that stereotypies are characteristic not only for OCD,
but also for some other psychiatric and neurological disorders (i.e.,
ASD and schizophrenia)45. The predictive validity of animal OCD
models is usually tested by serotonin reuptake inhibitors, which
ameliorate the symptoms. Examples of rodent OCD models include
Hoxb8 mutant mice, which display excessive self-grooming140,
5-HT(2C) receptor mutant mice with compulsive chewing141,
aromatase mutant mice with increased wheel-running and self-
grooming142, and Slitrk-5 mutant mice with abnormal striatum143
and excessive self-grooming45. Pharmacological modeling of OCD
usually involves serotonin and dopamine receptor modulation by
various substances, such as quinpirole139, 8-hydroxy-2-(di-ni-
polylamino)-tetralinehydrobromide144 and meta-chlorophenyl-
piperazine, which induce changes in rat spatial alternation that
can be used to assess the drugs’ efficacy145.
For zebrafish, the main approaches for modeling OCD symp-
toms have so far been genetic and pharmacological45. For example,
expression and function of the slitrk gene was characterized for
zebrafish146, which express eight orthologs of this gene—seven of
which guide neural system development—differentially at various
stages of development. However, Slitrk mutations are character-
istic not only for OCD, but also for myopia, schizophrenia and
Tourette syndrome, thereby necessitating a more full behavioral
characterization of Slitrk mutants. Quinpirole was investigated
for behavioral effects in zebrafish larvae, revealing an increased
locomotor activity, albeit it is unclear whether this phenotype nec-
essarily represents stereotypy147. Behavioral stereotypes (such as
tight circling and/or figure-8 patterns) can be observed in zebrafish
during psychostimulant drug testing, and may be considered as a
model of OCD behavior40,55. Another rodent test that may be appli-
cable to zebrafish is the T- or Y-maze spontaneous alternation145,
since animal models of OCD are expected typically to compulsively
prefer one arm over the other148.
General limitations of the zebrafish models
In order to emphasize the developing utility of a novel organism for
modeling complex human CNS deficits, it is best to provide a bal-
anced view of potential pros and cons of such models, with a sober
focus on their potential limitations. Some of the general key prob-
lems and challenges of disease modeling and treatment in animals
are summarized in Box 1. Other, more specific to zebrafish models,
are listed in Tab le 1, and will be discussed further. Indeed, despite
recent successes and rapid developments in the zebrafish CNS dis-
ease modeling field, there are still significant problems with these
models that must be taken into account, and may delay further
progress. Although some challenges have already been extensively
discussed in the literature15,20, it is important to keep discussing
them in order to find optimal solutions, and also to be better pre-
pared for the new challenges as the field develops.
As one example, genome duplication represents a problem for
some zebrafish models, because some genes with a single copy in
mammals may have two copies in zebrafish149–151. On the one
hand, this markedly increases the overall complexity of genetic
analyses necessary for correct data interpretation. On the other
hand, other researchers see opportunities in this genetic specificity,
because, for example, zebrafish models may help to study the effects
of genes whose null mutations lead to developmental lethality if
present in a single copy6. Related to genetic analyses is the problem
of a lack of a large number of available and characterized zebrafish
genetic strains, which stems not only from the relative novelty
of zebrafish in biomedical and neuroscience research15, but also
from rapid fertility loss owing to inbreeding in teleosts35. Currently,
there are no clear inbred lines in which all individuals are identi-
cal and homozygous35, and the level of genetic, physiological, and
behavioral characterization of these strains is not up to par with
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.
PERSPECTIVE
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384 Volume 46, No. 10 | OCTOBER 2017
the rich strain data available for laboratory rodents15. Notably,
near-homozygous zebrafish lines have been developed using heat
shock and early pressure, but have turned out to be too difficult to
maintain152, although ongoing investigations may soon help solve
this problem153.
Sex (gender) is widely recognized an important biological vari-
able to be factored into research designs, analyses, and reporting
in vertebrate animal and human studies. However, its status is
currently “complicated” for zebrafish models, since zebrafish have
no sex chromosomes, but a polygenic sex determination18,154,155.
As a result, for example, sex ratios in colonies usually vary, and
may often show no or aberrant sexual dimorphism in fish mod-
els compared to mammals18,154,155. Collectively, this may limit
the ability to access sex-dependent differences, often critical for
various human diseases. Additionally, under-reporting of sex
ratio determination in zebrafish studies reduces research repro-
ducibility and should be addressed by improving data reporting
standards in the field.
Other physiological differences may also pose a limitation to
be taken seriously. For example, there are species differences in
the blood-brain barrier, metabolism, thermoregulation, as well as
in the development, localization and function of neurotransmitter
signaling pathways, and brain morphology (e.g., cortex and hip-
pocampus)6,20. Furthermore, there are certain methodological
inconsistencies in zebrafish behavioral tests that may impact data
reproducibility156. For instance, the “test battery” effects remain
understudied, and, in turn, may affect detailed and consistent
descriptions of zebrafish neurophenotypes20. Moreover, despite
some progress in automation and recording equipment, there are
still technological difficulties in population-level analyses and
movement pattern recognition, especially evident in larvae stud-
ies29,157. Finally, there are also individual (i.e. set) and environ-
mental (i.e. setting) factors158 that may impact CNS models and
drug screens using zebrafish. Thus, further research is needed to
examine more thoroughly the role of sex, individual, strain and age
differences, as well as housing conditions, diet, testing time and
social status in zebrafish models159. Notably, there are currently no
community guidelines on a minimal set of extrinsic environmental
conditions which need to be presented in research publications to
ensure rigor, robustness and reproducibility of zebrafish experi-
ments. Collectively, this calls for further discussions among active
zebrafish researchers on how to best formulate, disseminate and
implement such guidelines, in order to markedly improve the col-
lection, organization, and sharing of zebrafish data.
In addition to these limitations of zebrafish models, there are also
some conceptual challenges (Tab le 1)20. For example, an important
question is how well does characterized zebrafish behavior match
up to other (e.g., rodent) models and humans? One can conclude
that, compared to what is known about human or mouse behavior,
relatively little literature exists on zebrafish behavior. Although this
is true at present, given the continued growth in zebrafish research,
such a gap may soon be filled by future research. For instance, some
behaviors important for CNS disease modeling are not found in
zebrafish40. However, spontaneous zebrafish behaviors are being
reported that show a striking similarity in many other domains
examined in mice and humans40.
Another current limitation for zebrafish, compared to human-
based research or rodent models, is the relatively small number
of well-established and validated zebrafish behavioral paradigms.
Again, this may be true simply because of the relatively recent adop-
tion of zebrafish as a model organism, rather than owing to any
major species differences in principal behavioral domains between
these vertebrate organisms. Indeed, those who consider fish very
different from humans should be reminded of a high genetic26,
physiological and behavioral40 homology between these species.
This, in our opinion, markedly overweighs the existing differences
that are conceptually less important from the point of view of the
models’ construct validity.
Conclusion
In summary, despite clear limitations of animal CNS disease models
(Box 1, Ta b le 1 ), mounting evidence summarized here empha-
sizes the tremendous potential of adult zebrafish for studying and
understanding human brain disorders. While some disorders, such
as anxiety and addiction, have already been extensively studied in
zebrafish, other CNS diseases, such as autism, ADHD and OCD,
merit further scrutiny and additional research efforts. The latter,
in turn, requires further support from the research community,
institutional administration, regulators, and research funders.
Importantly, although zebrafish research is expanding rapidly,
it is also not without stigmas, which continue to stifle innovation
in CNS disease modeling using this species6,11,20. One of the most
serious problems is the false perception of zebrafish—by both
researchers and regulators—as just “simpler mice, whose adop-
tion and use can lead to savings in money and lab space, but offer
little novel insights over more established rodent models. This
particular notion is, of course, both detrimental and misleading.
While there is nothing wrong with doing more research for less
time, space, and money, zebrafish research offers many other criti-
cal advantages discussed above, including serving as a unique ‘time
machine’ for rodent research validation, pushing back the natural
evolutionary clock. The ability to identify a shared system or path-
way affected in both rodent and zebrafish models, may eventually
lead to the discovery of a new core, fundamental mechanism of
CNS disease11,17,20.
Another widespread misconception is that zebrafish are only
good for genetics and developmental research. However, mounting
evidence discussed here suggests the opposite, demonstrating the
growing success of zebrafish models in almost every aspect of bio-
medicine, including modeling brain diseases15,16,28. Similarly, for
decades, research with larval zebrafish has historically dominated
the field. This situation has begun to change only recently, as more
and more laboratories are recognizing the value of adult zebrafish
models to study brain disorders, which represent the pathogenesis
of human adults15.
These short-sighted and dated stigmas continue to influence
the field, by not only discouraging innovative zebrafish research
and creating excessive regulatory obstacles, but also by prevent-
ing newcomers from joining this important area of translational
research. Clearly, it takes time to embrace a new model organism,
and perhaps even more time to apply it correctly in order to solve
the ‘big biomedical’ problems. For CNS disease modeling, we see
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.
PERSPECTIVE
LabAnimal Volume 46, No. 10 | OCTOBER 2017 385
the zebrafish tank half-full, and not half-empty. As this tank con-
tinues to fill, we view the existing knowledge gaps and limitations
as opportunities for strategic investment and growth, rather than as
warning signs to halt the advancement of innovative translational
research using zebrafish models, tests, and screens.
Received 13 April 2017; accepted 18 August 2017
Published online at http://www.nature.com/laban
ACKNOWLEDGMENTS
This research was supported by the Russian Foundation for Basic Research grant
16-04-00851 to A.V.K. He is the Chair of the International Zebrafish Neuroscience
Research Consortium (ZNRC), and current President of the International Stress
and Behavior Society (ISBS).
COMPETING FINANCIAL INTERESTS
The author declares no competing financial interests.
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Chronic psychosocial stress is increasingly recognized as a risk factor for late-onset Alzheimer's disease (LOAD) and associated cognitive deficits. Chronic stress also primes microglia and induces inflammatory responses in the adult brain, thereby compromising synapse-supportive roles of microglia and deteriorating cognitive functions during aging. Substantial evidence demonstrates that failure of microglia to clear abnormally accumulating amyloid-beta (Aβ) peptide contributes to neuroinflammation and neurodegeneration in AD. Moreover, genome-wide association studies have linked variants in several immune genes, such as TREM2 and CD33, the expression of which in the brain is restricted to microglia, with cognitive dysfunctions in LOAD. Thus, inflammation-promoting chronic stress may create a vicious cycle of aggravated microglial dysfunction accompanied by increased Aβ accumulation, collectively exacerbating neurodegeneration. Surprisingly, however, little is known about whether and how chronic stress contributes to microglia-mediated neuroinflammation that may underlie cognitive impairments in AD. This review aims to summarize the currently available clinical and preclinical data and outline potential molecular mechanisms linking stress, microglia and neurodegeneration, to foster future research in this field.
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