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The rights and wrongs of zebrafish: Behavioral phenotyping of zebrafish

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Animal models of disease are ultimately only as strong as the clinical phenotype(s) upon which they are based. Many obstacles impede our ability to design animal models of complex mental illnesses, such as depression. An animal model that attempts to re-create any disease strives to maximize construct, face, and predictive validities. Strategies to model depression in representative animals have largely focused on one or more symptoms of depression, which have left many knowledge gaps open. In approaching these knowledge gaps, there are three primary areas that we feel need to be focused on: development of translational animal models, identification of genetic determinants, and discovery of novel targets/biomarkers of depression. Here, we discuss how zebrafish may be utilized in the modeling and analysis of the mechanisms of depression. Furthermore, this chapter also provides a detailed description of the behavioral responses and makes recommendations for further development of these methods, and how they may be employed in forward genetic screening for mutations involved in depression-related phenotypes.
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33© Springer International Publishing Switzerland 2017
A.V. Kalueff (ed.), The rights and wrongs of zebrafish: Behavioral phenotyping
of zebrafish, DOI 10.1007/978-3-319-33774-6_2
Developing Zebrafish Depression-Related
Models
Julian Pittman and Angelo Piato
Abstract Animal models of disease are ultimately only as strong as the clinical
phenotype(s) upon which they are based. Many obstacles impede our ability to
design animal models of complex mental illnesses, such as depression. An animal
model that attempts to re-create any disease strives to maximize construct, face, and
predictive validities. Strategies to model depression in representative animals have
largely focused on one or more symptoms of depression, which have left many
knowledge gaps open. In approaching these knowledge gaps, there are three primary
areas that we feel need to be focused on: development of translational animal models,
identification of genetic determinants, and discovery of novel targets/biomarkers of
depression. Here, we discuss how zebrafish may be utilized in the modeling and
analysis of the mechanisms of depression. Furthermore, this chapter also provides a
detailed description of the behavioral responses and makes recommendations for
further development of these methods, and how they may be employed in forward
genetic screening for mutations involved in depression-related phenotypes.
Keywords Depression • Animal model development • Behavioral tests •
Endophenotypes • Pharmacological analysis
1 Introduction
To translate basic science lessons learned from animal models of depression to clinical
acumen, animal models of depression must be considered side-by-side with human
presentation of symptoms of illness. Modeling human depression (see further) in ani-
mals poses unique challenges given contributions from higher-order functions such as
J. Pittman (*)
Department of Biological and Environmental Sciences, Troy University,
McCall Hall Troy, AL 36083, USA
e-mail: jtpittman@troy.edu
A. Piato
Department of Pharmacology, Universidade Federal do Rio Grande do Sul,
Porto Alegre, Brazil
e-mail: angelopiato@ufrgs.br
34
emotions and cognitions to symptom presentations that are difficult, if not impossible
to pinpoint and study in animals. The foundation of research into the mechanisms of
depression must involve the development of novel behavioral paradigms, as they
allow the quantification of functional changes in the brain induced by mutations or
drugs, and will facilitate the discovery of underlying mechanisms and drug targets.
Depression is a common, serious and debilitating brain disorder [1]. Numerous
studies have examined the biological mechanisms of depression, and a considerable
amount of effort has been invested in the development of pharmacological treatments
[2–9]. For preclinical research, most of these studies have used rodents. Since a large
amount of data has been accumulated on rodent species, it may seem logical to think
that building upon this well-laid foundation is the only way to proceed. The abandon-
ment of rodent research is certainly not likely or recommended; however, utilization of
another vertebrate, zebrafish, appears to be a fruitful direction to pursue namely because
they are robust, small, reproduce quickly and possess evolutionarily conserved traits.
Zebrafish are showing promise as a model organism for experimental studies of
affective disorders [5, 10, 11]. This species is demonstrating the potential to be an
“exceptional” animal for investigating experimental, genetic, and pharmacological
models of neurobehavioral disorders, such as depression [5, 8, 12–18]. As a result of
the past three decades of intensive investigation with zebrafish, this species has
become geneticists’ favorite model organisms [16]. Zebrafish models strike an
optimal balance between system complexity and practical simplicity, possessing
brain anatomy, physiology, and genome very similar to those of other vertebrates
including mammals [19–25]. Furthermore, they are small, easy and cheap to maintain
in the laboratory, and are highly amenable to high-throughput screening (e.g., forward
genetic or drug screens). The latter is particularly noteworthy for the purposes of
unraveling the genetic, and in general the biological, mechanisms of complex brain
functions and the disorders of these functions. High-throughput screens may have the
ability to identify a significant proportion of the potentially large number of molecular
players involved in these functions [17, 26].
2 Pathogenesis of Depression and Model development
Depression remains a common disorder that affects approximately 15 million
Americans, despite the increasing knowledge on its pathophysiology and treatment
[19]. One of the obstacles is the lack of validated diagnostic tests based on biological
markers, which would allow us to predict treatment response in depressed patients.
Also, biomarkers that correlate to treatment response to antidepressants or
psychotherapy have not been identified so far. While imbalances in neurotransmitter
levels are certainly involved in the pathophysiology of depression, no single neu-
rotransmitter system is considered to be exclusively responsible. This is expected
considering the range of symptoms included in the depressive syndrome: depressed
mood, disinterest in usual activities, inability to feel pleasure, attention deficits,
sleep disturbances, appetite alterations, and suicidal ideation. A novel conceptual
approach is to consider depression as a systems-level ‘spectrum’ disorder that
J. Pittman and A. Piato
35
concerns several critical brain regions and connecting pathways. In order to enable
the development of scientifically-based rationales for innovative treatments, a
comprehensive understanding of the neurobiology of depression and its genetic and
environmental underpinnings is required.
The etiology of depression is currently viewed as a result of gene-environment
interactions that ultimately impact the three major monoamines—serotonin
(5-hydroxytryptamine, 5HT), norepinephrine (NE), and dopamine (DA). Recently
developed tools in molecular biology and brain imaging have provided further
evidence for the involvement of these neurotransmitter systems. Contrary to earlier
views [21], recent observations now support a preeminent role for central
dopaminergic circuits [27], which could explain the now well-reported suboptimal
response to selective serotonin reuptake inhibitors (SSRIs) and selective
serotonin- norepinephrine reuptake inhibitors (SNRIs).
Animal models cannot replicate the symptoms of depression in a complete manner,
since core symptoms of the disorder such as depressed mood, low self-esteem, or
suicidality are not possible to access in non-humans [25, 26, 28]. On the other hand,
there are depression endophenotypes that can be individually reproduced and evalu-
ated in animals [29]. Ideally, an animal model should represent a means to understand
the molecular, genetic, and epigenetic factors involved in the etiology of depression.
Animal models also afford insight into the pathology of depression by allowing us to
examine underlying molecular alterations and the causal relationship between genetic
or environmental factors, which are indispensable to develop novel therapies with
greater efficacy. The attempt to model a single symptom or endophenotype of a dis-
order, rather than to recapitulate its full phenotypic expression, is especially relevant
for medical disorders of unclear pathophysiology or genetic etiology, such as depres-
sion. For behavioral measures to be used as novel models they should meet reliability,
predictive, construct and face validity criteria as much as possible [23, 30].
3 Novel vs. Familiar, Open Field, Social Isolation Tests
Various methods have already been developed to induce and study depression- related
behaviors. Novelty is classically recognized as an anxiety-inducing factor in several
species, including humans. For instance, in the “open field test” rodents [31, 32] and
other animals, including fish [22, 33], are exposed to an unfamiliar (thus potentially
threatening) environment. The response to this novel environment is thought to be the
resultant of two opposing and conflicting tendencies: exploration, an active response
associated with the natural drive to explore unfamiliar places and objects, and anxiety,
a passive response associated with harm-avoidance Both behaviors are considered
adaptive, as exploratory activity may reveal food resources, mates and escape routes,
while passive anxiety-induced responses (immobility/freezing) may reduce predation
risk [32]. This interpretation may seem speculative, but quantitative genetic studies
point towards ambidirectional selection forces as the basis for open field behavior.
Thus, natural selection in rodents favored individuals that displayed intermediate
behaviors (not too active but not too passive either) [32], an observation that extends to
Developing Zebrafish Depression-Related Models
36
other vertebrates including fish [34]. This represents a particularly valuable application
for measuring depressive behavior in zebrafish and for identifying new genetic lines.
The evolutionary past of zebrafish is likely similar to that of mice and rats con-
sidering that zebrafish has also been under ambidirectional selection with regard to
behavioral responses induced by novelty. Therefore, when exposed to a novel envi-
ronment, zebrafish are expected to display moderate levels of anxiety-like behavior.
Importantly, behavioral experimentation generally includes animal handling by
humans, which also induces some level of anxiety. Analysis of novelty-induced
anxiety responses in zebrafish [35], demonstrate initially low levels of exploratory
activity that progressively increase across time. A typical “diving” response is
observed, i.e., increased amount of time spent on the bottom of the test tank, which
slowly decreases as the fish habituates to the novel environment [35] (see [22] for
similar findings). Nicotine was shown to have anxiolytic properties as this drug
reduced fear responses induced by novelty [35].
Decreased serotonergic activity is associated with depression and may be experi-
mentally induced by social isolation [36]. Specifically, rodents display hyperactive
and aggressive behavior following long-term social isolation, and anti-depressant
treatment is able to block these consequences [37]. Such isolation paradigms based
on serotonin deficits are used as experimental depression models in rodents [25],
and may be similarly employed with zebrafish.
4 Stress Models
Several protocols of unpredictable chronic stress (UCS) were reported to induce depres-
sion-like behavior in rodents [38–40]. These UCS models, however, are expensive,
time-consuming, long lasting (at least 4 weeks), and require a large physical infrastruc-
ture, besides presenting problems of reproducibility among laboratories [39, 41].
Although other labs [42, 43] investigated some aspects of stress in zebrafish,
ref. [44] was the first report to describe an experimental protocol to study the effects
of UCS in zebrafish. Compared to the most often used rodent protocols, a number
of advantages can be highlighted, such as low cost, ease of maintenance and manip-
ulation without the need for complex physical structure. In addition, while UCS
protocols are usually conducted over at least 4 weeks in rodents [39, 45], zebrafish
stressed during 7 or 14 days already showed behavioral, physiological and cellular
responses consistent with those observed in rodents and chronically stressed humans
[44]. The stress protocol induced anxiety, cognitive impairment and neuroendocrine
dysfunction, as measured by increased cortisol and CRF levels and decreased GR
expression. These results suggest that this model has adequate construct validity.
Subsequently, Chakravarty et al. [46] exposed zebrafish to a similar stress model
for 15 days. This protocol induced anxiety-like behavior and decreased neurogenesis.
The molecular markers corticotropin-releasing factor, calcineurin and phosphocyclic
AMP were altered. Moreover, using proteomics analyses, 18 proteins were found to
be modified in stressed-zebrafish, four of them (PHB2, SLC25A5, VDAC3 and
IDH2) related with mitochondrial viability.
J. Pittman and A. Piato
37
Another study [47] used a milder UCS protocol to study the effects of daytime
and nighttime stress on inhibitory avoidance learning, cortisol levels and gene
expression in Tuebingen zebrafish strain. Fish submitted to UCS displayed weaker
inhibitory avoidance learning compared to the control group. Regarding cortisol,
while fish submitted to 7 nights of UCS had higher levels of cortisol, no difference
was observed after 7 or 14 days of UCS. Important changes in bdnf, grα, grβ, grβ/
grα ratio, and mr genes were also observed after the 7-night UCS protocol.
In [48], the effects of a modified UCS protocol on molecular and physiologic
parameters related to stress response were assessed. Zebrafish submitted to UCS
protocol showed increase in cortisol levels and pro-opiomelanocortin, glucocorticoid
and mineralocorticoid receptors, prolactin, brain-derived neurotrophic factor,
hypocretin/orexin, and c-fos expression.
A recent study [49] also evaluated the effects of UCS on purinergic system in
zebrafish. UCS induced decrease in ecto-ADA (adenosine deaminase) and increases
in adenosine levels in zebrafish brain, without affect any ADA gene (ada1, ada2.1,
ada2.2, adaL, and adaasi) expression using quantitative reverse transcription. The
authors suggested that this increase in adenosine levels could help zebrafish to
achieve homeostasis during UCS. The UCS model in zebrafish remains to be more
fully pharmacologically validated, since its predictive validity was not assessed thus
far. Given the rich behavioral repertoire and the complex social interactions of indi-
viduals in a group, this model may contribute to a better understanding of the effects
of drugs modulating the stress axis (Table 1, Fig. 1).
Table 1 Main results of depression-related models in zebrafish
Model Main results References
Genetic Mutant grs357 HPA axis [50]
Blunted suppression of cortisol by dexamethasone
Spontaneous activity [51]
Stress Chronic stress Time in the tank bottom [44]
GR expression
Impaired memory
Cortisol and CRF expression
Time in the tank bottom [46]
Neurogenesis
Mitochondrial toxicity
Cortisol [47]
Altered BDNF, grα, grβ, grβ/grα ratio, and mr genes
Impaired memory
Cortisol levels [48]
POMC, GR, MR, prolactin, BDNF, hypocretin/
orexin, and c-fos expression
Adenosine [49]
Drug Reserpine Impaired locomotion [52]
Developing Zebrafish Depression-Related Models
38
5 Pharmacological Models for Depression-Like Responses
The motivation for the continued search for improved drugs to treat depression is
not only to improve the quality of life of those suffering from it, but also to aid in
our understanding of how depression develops, and what biological mechanisms
may underlie this disorder cluster. Another reason is that the currently available,
however numerous, drugs are often not efficacious or do not work for all patients.
One way zebrafish may be beneficial for such research is by speeding up the
discovery of the biological mechanisms responsible for the symptoms of depression.
This may be achieved using, for example, forward genetic screens that identify
mutations leading to the isolation of underlying genes. Another completely different
approach has been to search for compounds, or “small molecules”, which may alter
expression- like symptoms. It is thus important to consider what is known about the
psychopharmacological properties of zebrafish in the context of depression. For
example, can one consistently detect the efficacy of “gold standard” drugs for
depression using zebrafish? That is to say, does the zebrafish model have predictive
validity? Predictive validity is an important question for the use of novel model
organisms. The principal theme with regard to the translational relevance of
laboratory model organisms concerns the notion “evolutionary homology”, i.e.,
conservation of biological function across previously utilized species (e.g., rodents),
the novel laboratory species (e.g., zebrafish), and humans.
Many different pharmacological approaches can be employed to model
depression [53]. An example is the administration of psychostimulants, such as
amphetamine, which leads to hyperactivity and may be reversed by the administration
of anti-manic treatments, such as valproate. Additionally, repeated administration of
Fig. 1 Effects of different
manipulations on
behavioral, physiological
and molecular parameters
relevant to depression in
zebrafish
J. Pittman and A. Piato
39
psychostimulants induces a process of behavioral sensitization and may be used to
model bipolar disorder [24]. Considering that repeated exposure to cocaine can lead
to “cycling” in many neurochemical and physiological systems [54], bipolar-like
behavior could be replicated in zebrafish, for instance, by combining cocaine with
antipsychotic drugs. Another possibility is the induction of depressive-like behavior
due to withdrawal of an anxiolytic agent, such as ethanol; this protocol requires
chronic administration (minimum 3 weeks) of high doses of ethanol (1–3 %), and at
least 7 days post-withdrawal before behavioral symptoms are manifest. The SSRI
fluoxetine is able to reverse these depressive-like behaviors. In addition, quantita-
tive changes in immunoreactive neurons are observed following this protocol of
ethanol administration, mirroring many of the neurochemical findings of clinical
depression [53].
There is a great number of studies reporting the effects of ethanol exposure
across development in zebrafish. Findings comprise, for example, the strain-
dependent effect of developmental alcohol exposure [55], the long-term effects of
early embryonic ethanol exposure in adult animals [56], the development of adapta-
tion (tolerance) and withdrawal symptoms following chronic ethanol exposure [34,
57, 58], and numerous alterations induced by acute ethanol administration [58].
Importantly, the behavioral effects of ethanol depend on concentration and adminis-
tration regime, since lower doses of ethanol were shown to induce anxiolytic effects
(see [58] and [22]), while prolonged exposure and withdrawal was associated with
anxiogenic properties (see [57] and [22]). The behavioral effects induced by other
drugs of abuse have also been documented for zebrafish. Cocaine, for example, has
rewarding properties, and forward genetic screens have already been identified
zebrafish mutants with altered cocaine reinforced place preference in [59]. Similarly
to ethanol, also lead to anxiety/depression-related behaviors depending on drug
concentration and administration schedules [60, 61].
Classical anti-anxiety drugs have been shown to exhibit an anxiolytic profile in
zebrafish, such as fluromethylhistidine [62], benzodiazepines like diazepam, and the
widely prescribed SSRI fluoxetine, that decreases bottom-dwelling, erratic move-
ments, and whole-body cortisol levels [22], paralleling the responses observed in
rodents [63]. On the other hand, acute administration of drugs known to induce anxi-
ety in humans [64] and rodents [65], such as the benzodiazepine inverse agonist
FG-7142 [61] and caffeine [22], led to increased anxiety responses in zebrafish,
demonstrated by increased bottom-dwelling and erratic movements. Investigations
of stress hormone levels in zebrafish have revealed numerous similarities when com-
pared to the human stress response [5], strengthening the translational relevance of
zebrafish as a model organism in depression research. The sight of a predator, for
example, was shown to elevate cortisol levels in zebrafish [66]. It is important to
note that cortisol is the primary stress hormone of the hypothalamic-pituitary- adrenal
(HPA) axis in both human and zebrafish, but not in rodents, which use corticosterone
instead. At the Society for Neuroscience meeting in San Diego (2010), Baier and his
team demonstrated the generation of behavioral phenotypes resembling depression
by disrupting the zebrafish stress response [67]. Another study [50] found a mutation
in the glucocorticoid receptor gene in zebrafish that displayed depression-like
Developing Zebrafish Depression-Related Models
40
behaviors, suggesting that depression could be connected to an individual’s capability
to cope with stress. Furthermore, the SSRI fluoxetine (Prozac) ameliorated
depression-like behaviors is animals carrying the mutation. Molecules targeting the
glucocorticoid receptor and enhancing its activity instead of blocking it may lead to
promising novel therapies for the treatment of depression.
Also, depression-like motor retardation and social withdrawal have been reported
in adult zebrafish several days after exposure to reserpine [3]—a dopamine- depleting
drug known to elicit depression-like responses in rodents and clinical depression in
humans. However, with the use of all the above pharmacological treatments, one
must exercise extreme care and ensure there is some ability to provide a dissection
between anxiety and depression endpoints, especially given a high degree of comor-
bidity of anxiety with depression clinically. This may be achieved through careful
selection of pharmacological agents and behavioral tests (much development is
needed in this area), and confirmation of quantitative changes in neuronal circuits
involved in depression.
6 Model Limitations and Future Directions of Research
A significant difficulty with using zebrafish in depression research is the fact that
only recently the behavioral repertoire of this species has begun to be explored.
Although the number of behavioral studies published on zebrafish is on the rise
compared to classical laboratory species such as rat, mouse, or even the fruit fly,
zebrafish behavioral research is still in its infancy [28]. With the lack of reliable
behavioral tests and a thorough understanding of zebrafish behavioral features, the
behavioral and neurochemical consequences of gene mutation or drug exposure will
remain exceedingly difficult to study.
Given the complex mechanisms involved in the pathophysiology of depression,
one may assume the necessity of identifying a considerable number of molecular
players, i.e., genes and their protein products and the biochemical interactions
between the proteins. A possibility to tackle this complexity may be, at least initially,
to employ large scale screenings for mutations and drugs. This may result in the
identification of potential targets and leads that may be followed up on by more
targeted hypothesis-driven analyses. We are not, however, advocating large
screening as the only fruitful approach. A large number of mechanisms is awaiting
to be revealed, and “blind”, i.e., unbiased, screening applications may facilitate
their discovery. This is where zebrafish poses a major advantage over the classical
laboratory organisms.
The development of novel behavioral endpoints and observational methodolo-
gies, such as automated video-tracking systems, is important to reinforce the utility
of zebrafish as a model organism for depression research. The use of biomolecular
markers, such as gene and protein expression, to parallel zebrafish physiology with
behavioral data represents another critical research direction to pursue.
J. Pittman and A. Piato
41
References
1. Best JD, Alderton WK. Zebrafish: an in vivo model for the study of neurological diseases.
Neuropsychiatr Dis Treat. 2008;4(3):567–76.
2. Flint JSS. Animal models of psychiatric disease. Curr Opin Genet Dev. 2008;18:235–40.
3. Kyzar ER, Roth A, Gaikwad S, Green J, Collins C, El-Ounsi M, Davis A, Pham M, Stewart
AM, Cachat J, Zukowska Z, Kalueff AV. On making zebrafish sad and anxious: developing
novel aquatic models of affective disorders. IBNS Abstract, 2012.
4. Mathur P, Guo S. Use of zebrafish as a model to understand mechanisms of addiction and
complex neurobehaviroal phenotypes. Neurobiol Dis. 2010;40:66–72.
5. Alsop D, Vijayan MM. Development of the corticosteroid stress axis and receptor expression
in zebrafish. Am J Physiol Regul Integr Comp Physiol. 2008;294:711–9.
6. Alsop D, Vijayan M. The zebrafish stress axis: molecular fallout from the teleost specific
genome duplication event. Gen Comp Endocrinol. 2008;161:62–6.
7. Blaser R, Gerlai R. Behavioral phenotyping in zebrafish: comparison of three behavioral quan-
tification methods. Behav Res Methods. 2006;38:456–69.
8. Dooley K, Zon LI. Zebrafish: a model system for the study of human disease. Curr Opin Genet
Dev. 2000;10:252–6.
9. Norton W, Bally-Cuif L. Adult zebrafish as a model organism for behavioral genetics. BMC
Neurosci. 2010;11:90.
10. Sprague J, Doerry E, Douglas S, Westerfield M. The zebrafish information network (ZFIN): a
resource for genetic, genomic and developmental research. Nucleic Acids Res. 2001;29:
87–90.
11. Zon L, Peterson R. In vivo drug discovery in the zebrafish. Nat Rev Drug Discov.
2005;4:35–44.
12. Wullimann MF, Knipp S. Proliferation pattern changes in the zebrafish brain from embryonic
through early postembryonic stages. Anat Embryol. 2000;202:385–400.
13. Ward A, Lieschke G. The zebrafish as a model system for human disease. Front Biosci.
2002;7:d827–33.
14. Shin J, Fishman M. From zebrafish to human: modular medical models. Annu Rev Genomics
Hum Genet. 2002;3:311–40.
15. Moorman S. Development of sensory systems in zebrafish (Danio rerio). ILAR J. 2001;42:
292–8.
16. McGrath P, Li CQ. Zebrafish: a predictive model for assessing drug-induced toxicity. Drug
Discov Today. 2008;13:394–401.
17. Lele Z, Krone PH. The zebrafish as a model system in developmental, toxicological and trans-
genic research. Biotechnol Adv. 1996;14:57–72.
18. Blackburn J, Liu S, Raimondi A, Ignatius M, Salthouse C, Langenau D. High-throughput
imaging of adult fluorescent zebrafish with an LED fluorescence macroscope. Nat Protoc.
2011;6:229–41.
19. Kessler R, Chiu W, Demler O, Walters E. Prevalence, severity, and comorbidity of twelve-
month DSM-IV disorders in the National Comorbidity Survey Replication (NCS-R). Arch
Gen Psychiatry. 2005;62(6):617–27.
20. Willner P. The validity of animal models of depression. Psychopharmacology (Berl).
1984;83(1):1–16.
21. Nemeroff C. The neurobiology of depression. Sci Am. 1998;278:42–9.
22. Egan R, Bergner C, Hart P, Cachat J, Canavello P, Elegante M, Elkhayat S, Bartels B, Tien A,
Tien D, Mohnot S, Beeson E, Glasgow E, Amri H, Zukowska Z, Kalueff A. Understanding
behavioral and physiological phenotypes of stress and anxiety in zebrafish. Behav Brain Res.
2009;205:38–44.
23. Geyer M, Markou A. The role of preclinical models in the development of psychotropic drugs.
In: Davis K, Charney D, Coyle J, Nemeroff C, editors. Neuropsychopharmacology: the fifth
generation of progress. Philadelphia: Lippincott Williams & Wilkins; 2002. p. 446–55.
Developing Zebrafish Depression-Related Models
42
24. Kato T, Kubota M, Kasahara T. Animal models of bipolar disorder. Neurosci Biobehav Rev.
2007;31:832–42.
25. Leonard B. Animal models of depression. In: Briley M, Montgomery S, editors. Antidepressant
therapy. London: Martin Dunitz Ltd; 1998. p. 87–109.
26. Willner P. Animal models of depression: an overview. Pharmacol Ther. 1990;45:425–55.
27. Dunlop B, Nemeroff C. The role of dopamine in the pathophysiology of depression. Arch Gen
Psychiatry. 2007;64:327–37.
28. Weiss J, Kilts C. Animal models of depression and schizophrenia. Washington, DC: American
Psychiatric Press; 1995. p. 89–131.
29. Hasler G. Discovering endophenotypes for major depression. Neuropsychopharmacology.
2004;29:1765–81.
30. Geyer M, Markou A. Animal models of psychiatric disorders. In: Bloom F, Kupfer D, editors.
Psychopharmacology: the fourth generation of progress. New York: Raven; 1995. p. 787–98.
31. Prut L, Belzung C. The open field as a paradigm to measure the effects of drugs on anxiety-like
behaviors: a review. Eur J Pharmacol. 2003;463:3–33.
32. Crusio E, van Abeelen JH. The genetic architecture of behavioral responses to novelty in mice.
Heredity. 1986;56:55–63.
33. Csányi V, Gerlai R. Open-field behavior and the behavior-genetic analysis of the paradise fish
(Macropodus opercularis). J Comp Psychol. 1988;102:326–36.
34. Gerlai R, Csányi V. Genotype environment interaction and the correlation structure of behav-
ioral elements in paradise fish (Macropodus opercularis). Physiol Behav. 1990;47:343–56.
35. Levin E, Bencan Z, Cerutti D. Anxiolytic effects of nicotine in zebrafish. Physiol Behav.
2007;90:54–8.
36. Garattini S, Giacalone E, Valzelli L. Isolation, aggressiveness and brain 5-hydroxytriptamine
turnover. J Pharm Pharmacol. 1967;19:338–9.
37. Garzon J, del Rio J. Hypersensitivity induced in rats by long term isolation: further studies on
a new animal model for the detection of antidepressants. Eur J Pharmacol. 1981;74:287–94.
38. Mineur YS, Belzung C, Crusio WE. Effects of unpredictable chronic mild stress on anxiety
and depression-like behavior in mice. Behav Brain Res. 2006;175(1):43–50.
39. Willner P. Chronic mild stress (CMS) revisited: consistency and behavioural-neurobiological
concordance in the effects of CMS. Neuropsychobiology. 2005;52(2):90–110.
40. Yalcin I, Belzung C, Surget A. Mouse strain differences in the unpredictable chronic mild
stress: a four-antidepressant survey. Behav Brain Res. 2008;193(1):140–3.
41. Willner P. Validity, reliability and utility of the chronic mild stress model of depression: a
10-year review and evaluation. Psychopharmacology (Berl). 1997;134(4):319–29.
42. Cachat J, et al. Measuring behavioral and endocrine responses to novelty stress in adult zebraf-
ish. Nat Protoc. 2010;5(11):1786–99.
43. Champagne DL, et al. Translating rodent behavioral repertoire to zebrafish (Danio rerio): rel-
evance for stress research. Behav Brain Res. 2010;214(2):332–42.
44. Piato AL, et al. Unpredictable chronic stress model in zebrafish (Danio rerio): behavioral and
physiological responses. Prog Neuropsychopharmacol Biol Psychiatry. 2011;35(2):561–7.
45. Surget A, et al. Antidepressants recruit new neurons to improve stress response regulation.
Mol Psychiatry. 2011;16(12):1177–88.
46. Chakravarty S, et al. Chronic unpredictable stress (CUS)-induced anxiety and related mood
disorders in a zebrafish model: altered brain proteome profile implicates mitochondrial dys-
function. PLoS One. 2013;8(5):e63302.
47. Manuel R, et al. Unpredictable chronic stress decreases inhibitory avoidance learning in
Tuebingen long-fin zebrafish: stronger effects in the resting phase than in the active phase.
J Exp Biol. 2014;217(Pt 21):3919–28.
48. Pavlidis M, Theodoridi A, Tsalafouta A. Neuroendocrine regulation of the stress response in
adult zebrafish, Danio rerio. Prog Neuropsychopharmacol Biol Psychiatry. 2015;60:121–31.
49. Zimmermann FF, et al. Unpredictable chronic stress alters adenosine metabolism in zebrafish
brain. Mol Neurobiol. 2015;53:2518–28.
J. Pittman and A. Piato
43
50. Ziv L, et al. An affective disorder in zebrafish with mutation of the glucocorticoid receptor.
Mol Psychiatry. 2013;18(6):681–91.
51. Griffiths BB, et al. A zebrafish model of glucocorticoid resistance shows serotonergic modula-
tion of the stress response. Front Behav Neurosci. 2012;6:68.
52. Kyzar E, et al. Behavioral effects of bidirectional modulators of brain monoamines reserpine
and d-amphetamine in zebrafish. Brain Res. 2013;1527:108–16.
53. Pittman JT, Ichikawa KM. iPhone(R) applications as versatile video tracking tools to analyze
behavior in zebrafish (Danio rerio). Pharmacol Biochem Behav. 2013;106:137–42.
54. Antelman S, Caggiula A, Kucinski B, Fowler H, Gerhon S, Edwards D. The effects of lithium
on a potential cycling model of bipolar disorder. Prog Neuropsychopharmacol Biol Psychiatry.
1998;22:495–510.
55. Loucks E, Carvan 3rd MJ. Strain-dependent effects of developmental ethanol exposure in
zebrafish. Neurotoxicol Teratol. 2004;26:745–55.
56. Fernandes Y, Gerlai R. Long-term behavioral changes in response to early developmental
exposure to ethanol in zebrafish. Clin Exp Res. 2009;33:601–9.
57. Gerlai R, Chatterjee D, Pereira T, Sawashima T, Krishnannair R. Acute and chronic alcohol
dose: population differences in behavior and neurochemistry of zebrafish. Genes Brain Behav.
2009;8:586–99.
58. Gerlai R, Lahav M, Guo S, Rosenthal A. Drinks like a fish: Zebra fish (Danio rerio) as a behav-
ior genetic model to study alcohol effects. Pharmacol Biochem Behav. 2000;67:773–82.
59. Darland T, Dowling J. Behavioral screening for cocaine sensitivity in mutagenized zebrafish.
Proc Natl Acad Sci U S A. 2001;98:11691–6.
60. Ninkovic J, Bally-Cuif L. The zebrafish as a model system for assessing the reinforcing prop-
erties of drugs of abuse. Methods. 2006;39:262–74.
61. Lopez-Patino M, Yu L, Cabral H, Zhdanova I. Anxiogenic effects of cocaine withdrawal in
zebrafish. Physiol Behav. 2008;93:160–71.
62. Peitsaro N, Kaslin J, Anichtchik O, Panula P. Modulation of the histaminergic system and
behaviour by alpha-fluoromethylhistidine in zebrafish. J Neurochem. 2003;86:432–41.
63. Dulawa S, Holick K, Gundersen B, Hen R. Effects of chronic fluoxetine in animal models of
anxiety and depression. Neuropsychopharmacology. 2004;29:1321–30.
64. Childs E, Hohoff C, Deckert J, Xu K, Badner J, de Wit H. Association between ADORA2A
and DRD2 polymorphisms and caffeine-induced anxiety. Neuropsychopharmacology.
2008;33:2791–800.
65. El Yacoubi M, Ledent C, Parmentier M, Costentin J, Vaugeois J. The anxiogenic-like effect of
caffeine in two experimental procedures measuring anxiety in the mouse is not shared by
selective A(2A) adenosine receptor antagonists. Psychopharmacology (Berl).
2000;148:153–63.
66. Barcellos G, Ritter F, Kreutz C, Quevedo M, Bolognesi da Silva L, Bedin C. Whole-body
cortisol increases after direct and visual contact with a predator in zebrafish, Danio rerio.
Aquaculture. 2007;272:774–8.
67. Baier H. Depression-like behavior in zebrafish mutants with disruption of the glucocorticoid
receptor. Society for Neuroscience Annual Meeting, 2010. Abstract 884.1.
Developing Zebrafish Depression-Related Models
http://www.springer.com/978-3-319-33773-9
... Zebrafish models are also receiving the growing recognition as a useful tool for studying various psychiatric illnesses, including psychotic [33,34], affective [35,36], neurodevelopmental [37], social [38], eating [39] and other brain disorders [23]. Collectively, this raises the possibility that zebrafish can be a valuable model system to study mood disorders, including depression [40,41] and its critical hallmark, despair. Here, we critically discuss the developing potential and important translational implications of zebrafish models for studying despair and its mechanisms, and overview the utility of such aquatic models and tests for antidepressant drug screening. ...
... In general, modeling depression in zebrafish faces several challenges, including creating valid, translational models of this disorder, identifying genetic and molecular biomarkers of depression, and improving antidepressant drug screening and discovery [40]. Can zebrafish J o u r n a l P r e -p r o o f offer good models for despair as a critical symptom of depression? ...
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Depression is a widespread and severely debilitating neuropsychiatric disorder whose key clinical symptoms include low mood, anhedonia and despair (the inability or unwillingness to overcome stressors). Experimental animal models are widely used to improve our mechanistic understanding of depression pathogenesis, and to develop novel antidepressant therapies. In rodents, various experimental models of 'behavioral despair' have already been developed and rigorously validated. Complementing rodent studies, the zebrafish (Danio rerio) is emerging as a powerful model organism to assess pathobiological mechanisms of depression and other related affective disorders. Here, we critically discuss the developing potential and important translational implications of zebrafish models for studying despair and its mechanisms, and the utility of such aquatic models for antidepressant drug screening.
... There are several behavioral protocols extensively used and described for this species, such as the novel tank and light/dark tests. The novel tank diving test is based on an anti-predatory defense mechanism that induces fish to swim at the bottom of the tank, whereas the light/dark test evaluates anxiety based on the innate preference of adult zebrafish to dark over light areas (Levin, Bencan & Cerutti, 2007;Gebauer et al., 2011;Khan et al., 2017;Pittman & Piato, 2017). ...
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Studies have suggested that oxidative stress may contribute to the pathogenesis of mental disorders. In this context, molecules with antioxidant activity may be promising agents in the treatment of these deleterious conditions. Acetyl-L-carnitine (ALC) is a multi-target molecule that modulates the uptake of acetyl-CoA into the mitochondria during fatty acid oxidation, acetylcholine production, protein, and membrane phospholipid synthesis, capable of promoting neurogenesis in case of neuronal death. Moreover, neurochemical effects of ALC include modulation of brain energy and synaptic transmission of multiple neurotransmitters, including expression of type 2 metabotropic glutamate (mGlu2) receptors. The aim of this study was to investigate the effects of ALC in zebrafish by examining behavioral and biochemical parameters relevant to anxiety and mood disorders in zebrafish. ALC presented anxiolytic effects in both novel tank and light/dark tests and prevented the anxiety-like behavior induced by an acute stressor (net chasing). Furthermore, ALC was able to prevent the lipid peroxidation induced by acute stress in the zebrafish brain. The data presented here warrant further investigation of ALC as a potential agent in the treatment of neuropsychiatric disorders. Its good tolerability also subsidizes the additional studies necessary to assess its therapeutic potential in clinical settings.
... Current modeling of depression in zebrafish has largely focused on one or more symptoms of this disorder (Table 1), aiming to: i) develop translational zebrafish models, ii) identify genetic determinants and novel biomarkers of depression, and iii) improve antidepressant drug discovery (Pittman & Piato, 2017). For example, the learned helplessness (LH) paradigm recently developed in zebrafish (do Nascimento, et al., 2016), has been conceptually adapted from rodent LH models that pre-expose animals to uncontrollable unescapable events (e.g., foot or tail shock). ...
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Depression is a wide-spread, debilitating psychiatric disorder. Mainly rodent-based, experimental animal models of depression are extensively used to probe the pathogenesis of this disorder. Here, we emphasize the need for innovative approaches to studying depression, and call for a wider use of novel model organisms, such as the zebrafish (Danio rerio). Highly homologous to humans and rodents, zebrafish are rapidly becoming a valuable tool in translational neuroscience research, but have only recently been utilized in depression research. Multiple conceptual and methodological problems, however, arise in relation to separating putative zebrafish depression-like states from motor and social deficits or anxiety. Here, we examine recent findings and the existing challenges in this field, to encourage further research and the use of zebrafish as novel organisms in cross-species depression modeling.
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There is accumulating evidence on the use of N-acetylcysteine (NAC) in the treatment of patients with neuropsychiatric disorders. As a multi-target drug and a glutathione precursor, NAC is a promising molecule in the management of stress-related disorders, for which there is an expanding field of research investigating novel therapies targeting oxidative pathways. The deleterious effects of chronic stress in the central nervous system are a result of glutamatergic hyperactivation, glutathione (GSH) depletion, oxidative stress, and increased inflammatory response, among others. The aim of this study was to investigate the effects of NAC in zebrafish submitted to unpredictable chronic stress (UCS). Animals were initially stressed or not for 7 days, followed by treatment with NAC (1 mg/L, 10 min) or vehicle for 7 days. UCS decreased the number of entries and time spent in the top area in the novel tank test, which indicate increased anxiety levels. It also increased reactive oxygen species (ROS) levels and lipid peroxidation (TBARS) while decreased non-protein thiols (NPSH) and superoxide dismutase (SOD) activity. NAC reversed the anxiety-like behavior and oxidative damage observed in stressed animals. Additional studies are needed to investigate the effects of this agent on glutamatergic modulation and inflammatory markers related to stress. Nevertheless, our study adds to the existing body of evidence supporting the clinical evaluation of NAC in mood disorders, anxiety, post-traumatic stress disorder, and other conditions associated with stress.
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Zebrafish (Danio rerio Hamilton) are increasingly used as model to study effects of chronic stress on brain and behaviour. In rodents unpredictable chronic stress (UCS) has a stronger effect on physiology and behaviour during the active phase than the resting phase. Here, we applied UCS during day-time (active phase) for 7 and 14 days or during the night-time (resting phase) for 7 nights in an in-house reared Tuebingen Long-Fin (TLF) zebrafish strain. Following UCS, inhibitory avoidance learning was assessed using a 3-day paradigm where fish learn to avoid swimming from a white to a black compartment where they will receive a 3V shock. Latencies of entering the black compartment were recorded before training (day 1; first shock) and after training on day 2 (second shock) and day 3 (no shock, tissue sampling). Fish were sacrificed to quantify whole-body cortisol content and expression levels of genes related to stress, fear and anxiety in the telencephalon. Following 14 days UCS during the day, inhibitory avoidance learning decreased (lower latencies on day 2 and 3); minor effects were found following 7 days UCS. Following 7 nights UCS inhibitory avoidance learning decreased (lower latency on day 3). Whole-body cortisol levels showed a steady increase compared to controls (100%) from 7 days UCS (139%), 14 days UCS (174%l) to 7 nights UCS (231%), suggestive of an increasing stress load. Only in the 7 nights UCS group expression levels of corticoid receptor genes (mr, gr-alpha, gr-beta) and of bdnf were increased. These changes are discussed as adaptive mechanisms to maintain neuronal integrity and prevent overload, and indicative of a state of high stress load. Overall, our data suggest that stressors during the resting phase have a stronger impact than during the active phase. Our data warrant further studies on the effect of UCS on stress-axis related genes, especially gr-beta; in mammals this receptor has been implicated in glucocorticoid resistance and depression.
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Errors in Byline, Author Affiliations, and Acknowledgment. In the Original Article titled “Prevalence, Severity, and Comorbidity of 12-Month DSM-IV Disorders in the National Comorbidity Survey Replication,” published in the June issue of the ARCHIVES (2005;62:617-627), an author’s name was inadvertently omitted from the byline on page 617. The byline should have appeared as follows: “Ronald C. Kessler, PhD; Wai Tat Chiu, AM; Olga Demler, MA, MS; Kathleen R. Merikangas, PhD; Ellen E. Walters, MS.” Also on that page, the affiliations paragraph should have appeared as follows: Department of Health Care Policy, Harvard Medical School, Boston, Mass (Drs Kessler, Chiu, Demler, and Walters); Section on Developmental Genetic Epidemiology, National Institute of Mental Health, Bethesda, Md (Dr Merikangas). On page 626, the acknowledgment paragraph should have appeared as follows: We thank Jerry Garcia, BA, Sara Belopavlovich, BA, Eric Bourke, BA, and Todd Strauss, MAT, for assistance with manuscript preparation and the staff of the WMH Data Collection and Data Analysis Coordination Centres for assistance with instrumentation, fieldwork, and consultation on the data analysis. We appreciate the helpful comments of William Eaton, PhD, Michael Von Korff, ScD, and Hans-Ulrich Wittchen, PhD, on earlier manuscripts. Online versions of this article on the Archives of General Psychiatry Web site were corrected on June 10, 2005.
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Stress is considered a risk factor for several human disorders. Despite the broad knowledge of stress responses in mammals, data on the relationship between unpredictable chronic stress (UCS) and its effects on purinergic signaling are limited. ATP hydrolysis by ectonucleotidases is an important source of adenosine, and adenosine deaminase (ADA) contributes to the control of the nucleoside concentrations. Considering that some stress models could affect signaling systems, the objective of this study was to investigate whether UCS alters ectonucleotidase and ADA pathway in zebrafish brain. Additionally, we analyzed ATP metabolism as well as ada1, ada2.1, ada2.2, adaL, and adaasi gene expression in zebrafish brain. Our results have demonstrated that UCS did not alter ectonucleotidase and soluble ADA activities. However, ecto-ADA activity was significantly decreased (26.8 %) in brain membranes of animals exposed to UCS when compared to the control group. Quantitative reverse transcription PCR (RT-PCR) analysis did not show significant changes on ADA gene expression after the UCS exposure. The brain ATP metabolism showed a marked increase in adenosine levels (ADO) in animals exposed to UCS. These data suggest an increase on extracellular adenosine levels in zebrafish brain. Since this nucleoside has neuromodulatory and anxiolytic effects, changes in adenosine levels could play a role in counteracting the stress, which could be related to a compensatory mechanism in order to restore the homeostasis.
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Brain monoamines play a key role in the regulation of behavior. Reserpine depletes monoamines, and causes depression and hypoactivity in humans and rodents. In contrast, d-amphetamine increases brain monoamines' levels, and evokes hyperactivity and anxiety. However, the effects of these agents on behavior and in relation to monoamine levels remain poorly understood, necessitating further experimental studies to understand their psychotropic action. Zebrafish (Danio rerio) are rapidly emerging as a promising model organism for drug screening and translational neuroscience research. Here, we have examined the acute and long-term effects of reserpine and d-amphetamine on zebrafish behavior in the novel tank test. Overall, d-amphetamine (5 and 10mg/L) evokes anxiogenic-like effects in zebrafish acutely, but not 7 days later. In contrast, reserpine (20 and 40mg/L) did not evoke overt acute behavioral effects, but markedly reduced activity 7 days later, resembling motor retardation observed in depression and/or Parkinson's disease. Three-dimensional 'temporal' (X, Y, Time) reconstructions of zebrafish locomotion further supports these findings, confirming the utility of 3D-based video-tracking analyses in zebrafish models of drug action. Our results show that zebrafish are highly sensitive to drugs bi-directionally modulating brain monoamines, generally paralleling rodent and clinical findings. Collectively, this emphasizes the potential of zebrafish tests to model complex brain disorders associated with monoamine dysregulation.