Genes, Brain and Behavior (2004) 3: 6374

Copyright

Blackwell Munksgaard 2003

Review

Linking genes to brain, behavior and neurological

diseases: what can we learn from zebrafish?
S. Guo
Department of Biopharmaceutical Sciences, Programs in Human
Genetics, Biological Sciences, and Neuroscience, Wheeler
Center for the Neurobiology of Addiction, University of California,
San Francisco, CA, USA
*Corresponding author: S. Guo, Department of Biopharmaceutical
Sciences, Programs in Human Genetics, Biological Sciences,
and Neuroscience, Wheeler Center for the Neurobiology
of Addiction, University of California, San Francisco, CA
941430446, USA. E-mail: suguo@itsa.ucsf.edu

How our brain is wired and subsequently generates

functional output, ranging from sensing and locomotion
to emotion, decision-making and learning and memory,
remains poorly understood. Dys-regulation of these processes can lead to neurodegenerative, as well as neuropsychiatric, disorders. Molecular genetic together with
behavioral analyses in model organisms identify genes
involved in the formation of neuronal circuits, the execution of behavior and mechanisms involved in neuropathogenesis. In this review I will discuss the current
progress and future potential for study in a newly established vertebrate model organism for genetics, the zebrafish Danio rerio. Where available, schemes and results
of genetic screens will be reviewed concerning the sensory, motor and neuromodulatory monoamine systems.
Genetic analyses in zebrafish have the potential to provide important insights into the relationship between
genes, neuronal circuits and behavior in normal as well
as diseased states.
Keywords: Anxiety, behavior, dopamine, fear, goal-directed
behavior, monoamine system, motor system, neuronal circuitry, reward, sensory system, serotonin, zebrafish
Received 4 August 2003, revised 3 September 2003,
accepted for publication 23 September 2003

The goal of this review is to acquaint the reader with the

newly established vertebrate model organism for genetics,
the zebrafish Danio rerio, and the opportunity it provides us
to understand the molecular and cellular mechanisms of
behavior and behavioral disorders. I will start by presenting
the salient features of zebrafish for genetic study, the powerdoi: 10.1046/j.1601-183X.2003.00053.x

ful molecular genetic tools developed in zebrafish, and the

impact of sequencing the whole genome. I will go on to
discuss the use of zebrafish to study sensory perception
and locomotor regulation by outlining the behavioral assays
developed for large-scale genetic screening. I will focus on
several key mutants that affect either the development or
function of specific neuronal circuits involved in the behavior.
These parts will be brief, as they have been recently
reviewed elsewhere (Baier 2000; Drapeau et al. 2002;
Goldsmith & Harris 2003; Kratz et al. 2002; Li 2001;
Neuhauss 2003; Whitfield 2002). Finally, I will discuss the
use of zebrafish to study more complex behaviors including
goal-directed behavior (motivation and reward), emotions
(anxiety and fear), learning and memory and the role of
brain monoamine systems in modulating these behaviors.

Zebrafish: a new vertebrate genetic

model organism for understanding neural
development, function and disease
Zebrafish, a freshwater tropical teleost fish common to most
pet stores, was first chosen for laboratory study by the late
George Streisinger about 30 years ago. His vision was to
apply molecular genetics to unravel neural development
down to single cell and single molecule in a vertebrate (for
a review of detailed historical perspective, see Grunwald &
Eisen 2002). Why did Streisinger choose zebrafish? The
reasons are quite obvious today: zebrafish is a diploid vertebrate with a good balance of complexity and simplicity. It is a
small creature of only 35 cm in length, and reproduces
robustly. These properties make it easy to maintain a large
number of animals in a relatively small space, a prerequisite
for carrying out large-scale genetic study. Such a prerequisite
is found in invertebrate model organisms such as Caenorhabditis elegans and Drosophila, but is lacking in vertebrate
model organisms such as mice. The embryo and larva of
zebrafish are transparent and develop amazingly rapidly: in
merely 5 days, swimming, self-feeding larvae, also known
as fry, emerge from fertilized zygotes. This entire process
unravels in a Petri dish (Fig. 1).
Since the introduction of zebrafish into the laboratory,
many milestones have been achieved that firmly establish
zebrafish as a prominent genetic model organism for biology
and medicine. Streisingers pioneering work on establishing

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1hour

2-day

3-month

5-day

Figure 1: The life cycle of zebrafish. Hundreds of eggs can be

produced from a single mating. A typical vertebrate body plan is
laid out by 2 days post fertilization. By 5 days post fertilization, the
larval zebrafish are free-living, hunt for food and escape from
predators. By three months post fertilization, zebrafish are ready
for reproduction.

homozygous diploid clones and lethal-free strains (Streisinger

et al. 1981), and pilot mutagenesis screening (Walker &
Streisinger 1983), laid an important foundation for later
large-scale genetic screens. Therein 4000 embryonic lethal
mutations were identified that affect virtually all aspects of
visible developmental processes (Driever et al. 1996; Haffter
et al. 1996). In addition to genetics, cell biological and anatomical studies uncovered the segmental structure of the brain
and identified the role of cell lineage in generating diverse
groups of neurons (Kimmel 1993; Kimmel et al. 1990).
The goal of forward genetic screens is to reveal the molecular identity of genes affected by mutations (Fig. 2). Genomic

technologies such as RAPD (Rapid Amplification of

Polymorphic DNA) and AFLP (Amplified Fragment Length
Polymorphism) have been used to positionally clone genes
defined by mutations (Donovan et al. 2000; Guo et al. 2000;
Zhang et al. 1998). A genetic linkage map composed of more
than 4000 polymorphic microsatellite markers (Knapik et al.
1998) and two radiation hybrid maps composed of both
microsatellite markers and ESTs (Expressed Sequence
Tags) (Geisler et al. 1999; Hukriede et al. 1999) have been
constructed. A syntenic relationship between zebrafish and
mammalian species has been determined (Barbazuk et al.
2000; Woods et al. 2000). Recently, rapid mapping of zebrafish mutations with SNPs (Single Nucleotide Polymorphisms)
and oligonucleotide microarrays has also been developed
(Stickney et al. 2002). These technological advancements
greatly facilitate gene identification from mutations. In addition, tools for insertional mutagenesis have been established
to isolate insertional mutants that could be rapidly cloned
(Amsterdam et al. 1999). Complementing forward genetic
analysis, reverse genetic methods are also in place. These
include the morpholino antisense knockdown, which is a
simple method to inactivate known genes in early embryos
(Nasevicius & Ekker 2000). Recently, TILLING (TargetedInduced Local Lesions IN Genomes) (McCallum et al.
2000), a method originally developed in Arabidopsis, has
been successfully adapted to zebrafish (Wienholds et al.
2002). Mutations ranging from null (complete loss of function) to hypomorphic (partial loss-of-function) alleles can be
isolated in virtually any gene of interest (Fig. 3). Finally, both
forward and reverse genetic studies in zebrafish will be
greatly facilitated by the ongoing Sanger Center zebrafish
genome sequencing project, which is due to be completed
in the year 2003. Sequencing data are freely available in
GenBank as well as at the Sanger Center web site and a
centralized web-based database (ZFIN) (Sprague et al. 2001).

Figure 2: Schemes for forward

genetic screens. Adult male zebrafish
are mutagenized with the chemical
mutagen ENU (ethyl nitrosourea). ENU
induces mutations in the spermatogonia.
Mutagenized males are mated with
wildtype females to produce the F1
generation. The F1 generation is heterozygous for any induced mutations. Thus,
only dominant mutations can be
recovered if genetic screens are
conducted on the F1 generation. To
identify recessive mutations, F1 fish
need to be crossed to obtain F3
generations, in which homozygous
mutations are present (two-generation
screen). Alternatively, F1 female fish can
be induced to undergo gynogenesis by
applying early pressure (EP); therefore,
homozygous mutations can be recovered
in the F2 generation.

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Genes, Brain and Behavior (2004) 3: 6374

Genes, circuits and behavior in zebrafish

Figure 3: The scheme of the reverse genetic method,

TILLING. Similar to a forward genetic screen, the F1
generation that is heterozygous for any induced mutations is
raised to adulthood. A frozen sperm library is from F1 fish. In
addition, genomic DNA is prepared from individual F1 fish to
establish a corresponding genomic DNA library. To identify a
mutation in a particular gene of interest, gene-specific primers
are used to amplify PCR products from the genomic DNA library.
PCR products are digested with CEL1 enzyme, which cuts at
mismatches. Gel electrophoresis is carried out to identify which
F1 carries a mutation in the gene of interest. Once the F1
carrying a mutation in the gene of interest is identified, the line
can be recovered from the frozen sperm library.

Taken together, zebrafish hold great potential for studying

developmental processes, organ function and human diseases (Dooley & Zon 2000; Shin & Fishman 2002).
Zebrafish are highly suitable for studying behavior to elucidate the role of genes in the formation and function of
neuronal circuitry. Zebrafish embryos (05 days old) already
exhibit simple sensory and locomotor abilities. Zebrafish
larvae (5 days to 2-weeks old) are free-living and need to hunt
for food and escape from predators, thus they possess
many patterns of behavior. Yet both embryos and larvae
are optically transparent, making them highly accessible to
cellular study of neuronal circuitry. In fact, single neuronal
connection or activity can be directly visualized with the
utilization of fluorescent proteins, and single neuron ablation
can also be achieved with a laser (Fetcho & Liu 1998).
Genes themselves do not generate behavior. Rather,
genes control the development and function of neuronal
Genes, Brain and Behavior (2004) 3: 6374

circuits, which produce behavior. In general, genetic analysis

of behavior is expected to identify two classes of mutations:
one affects the formation (development) of neuronal circuits,
and the other alters their function. Both classes of mutations,
when specific enough, will provide important insights into
the neuronal circuitry that underlie behavior. Therefore, in the
following paragraphs, both classes of mutations will be discussed in the context of specific behavior.
It is also important to keep in mind that the expressivity or
function of genes, as well as the connectivity patterns of
neuronal circuits, can be modified by the external environment, leading to behavioral variability and plasticity (Crabbe
et al. 1999). In addition, genetic background can also have a
strong influence on behavior (Gerlai 1996). Therefore, when
designing behavioral assays, the factors of environment and
genetic background should be taken into consideration.
Although an ideal behavioral assay that is suitable for largescale genetic screens should be robust enough to show
minimal sensitivity to these factors, such an assay is not
always obtainable. Therefore, genetic background and environmental factors need to be tightly monitored and controlled
during the study of behavior. In zebrafish, several strains of
different genetic background are available. AB is a laboratory
strain that has been bred for many generations in the
Oregon lab and may be a good choice for behavioral study.
Other strains include Wik, Tu, TL and India (Johnson & Zon
1999). These strains show high interstrain genetic
polymorphisms, and therefore are likely to exert different
behavior.
In summary, the zebrafish offers a unique advantage for
large-scale genetic screens to be carried out in a vertebrate.
Compared to Drosophila and C. elegans, the nervous system
of zebrafish is more comparable to that of humans.
Compared to mice, zebrafish is more amenable to forward
genetic study, which has proven difficult and costly to carry
out in mice. In the following paragraphs I will discuss the
behavioral genetic study to understand multiple sensory
systems, the motor systems and the neuromodulatory
monoamine systems. Behaviors that are largely reflexive in
nature are considered relatively simple behaviors, whereas
non-reflexive behaviors, which are generally termed here as
goal-directed behaviors, are considered more complex
behaviors.

Vision, olfaction and mechanosensory

transduction
Contact with the external environment is through the sensory systems, including eyes, nose, ears and peripheral
nerves. Sensing the surroundings is the first step toward
the generation of complex behavior. Studies in zebrafish
are providing considerable insights into the formation of sensory circuits, and are holding great potential for elucidating
the function of sensory systems at an organismal level.

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The visual system

Among all the sensory systems in zebrafish, the visual system is the best studied, due to its great accessibility.
Mutants that affect the development as well as function of
the visual system have been isolated through both morphological and behavioral screens (Baier et al. 1996; Brockerhoff
et al. 1995; Li & Dowling 1997; Malicki et al. 1996a;
Neuhauss et al. 1999).
An interesting observation was made that blind fish tend
to have a darker appearance compared to normal fish when
placed in a light background (Neuhauss et al. 1999). This
appearance is particularly easy to see in larval zebrafish. As
for many lower vertebrates, zebrafish possess the ability to
change their appearance as a means of environmental adaptation. That is, the melanin pigments (melanosomes) in their
pigment cells (melanophores) disperse or aggregate in
response to the intensity of light. This cellular behavior is
mediated by retinal-hypothalamic projections, which in turn
regulate the secretion of two pituitary hormones that control
melanosome movements (Balm & Gronevald 1998). Because
of the inability to sense light, blind fish adapt to have a dark
appearance. This simple assay can be used as a primary
screening method for visual impairment in larval zebrafish.
In addition to screening for dark fish, two other robust and
perhaps more specific and quantifiable assays for vision are
the optokinetic response (OKR) and optomotor response
(OMR), which are largely applied to larval zebrafish. In the
OKR assay, the fish are partially immobilized with methyl
cellulose in a Petri-dish, which is placed inside a rotating
drum. A smooth pursuing eye movement in the direction of
the moving objects followed by a rapid saccadic eye movement is consistently observed in normal fish (Clark 1981). In
the OMR assay, fish swim in the direction of the moving
objects when placed in a circular container surrounded with
rotating black and white stripes (Clark 1981). Recently,
computer-generated images (black and white stripes) are
presented below a chamber containing freely moving larval
zebrafish. It is observed that larval fish also swim in the
direction of the moving stripes. As a consequence, fish congregate towards the other end of the chamber (Neuhauss
et al. 1999). Both OKR and OMR are robust, i.e. 90% fish
will respond to the stimuli; therefore, these assays are highly
suitable for genetic screens.
A behavioral test based on the visually mediated escape
response has been developed to quantitatively measure
visual sensitivity in adult zebrafish (Li & Dowling 1997). The
adult fish is confined to a circular container with a pole in the
center. Upon visually encountering a black segment rotating
outside the container (serving as a threatening object), the
fish immediately turn and rapidly swim away from the black
segment. 8090% of time the fish will respond to the
stimuli in a positive way (i.e. an escape response is evoked).
These behavioral assays share simple and robust features.
Genetic screens have been conducted using these assays. A

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number of interesting mutants have been isolated. Among

them, a mutation named lakritz (lak) was originally identified
to have a dark appearance (Kelsh et al. 1996), and later found
to be defective in both OMR and OKR assays (Neuhauss
et al. 1999). The behavioral defect of lak is caused by a
specific developmental deficit of retinal ganglion neurons
due to a mutation in the ath5 gene, a vertebrate homologue
of the Drosophila bHLH gene atonal (Kay et al. 2001). In
Drosophila, the atonal gene is required for the development
of photoreceptors in the compound eye and peripheral
chordotoal organs (Jarman et al. 1993; Jarman et al. 1994).
In vertebrates including zebrafish, xenopus and mice, ath5 is
required to determine retinal ganglion neuron differentiation.
Although carrying out seemingly different functions in Drosophila and vertebrates, the biochemical mechanism of
action of the atonal/ath5 gene appears to be highly conserved. Another mutant (belladonna) displays an OKR behavior that is directionally reversed. Cellular study showed that
optic chiasm failed to form in the mutant, suggesting the
possible underlying circuitry defect (Rick et al. 2000). In
addition to larval stage-based screens, a genetic screen in
adult zebrafish has identified dominant mutations that lead to
night blindness (nb) (Li & Dowling 1997). Heterozygous nba
and nbb mutations have specific visual defects due to adult
onset retinal neurodegeneration, whereas homozygous
mutations for both loci are embryonic lethal due to widespread neurodegeneration.
Can zebrafish mutations be useful to understand human
visual disorders? The phenotypes of mutant zebrafish have
shown resemblances to certain conditions of human retinal
disorders. For example, the nagie oko mutant, which has an
eye and brain patterning defect, showed a similarity to the
oculo-cerebro-renal human disorder and encoded a membrane-associated guanylate kinase (Wei & Malicki 2002).
This suggests that the studies of zebrafish mutations might
provide insights into human retinal neurodegeneration.

The olfactory system

Besides vision, another way to sense the environment is
through smell. Olfactory perception involves odorant interaction
with receptors that are expressed on the surface of odorant receptor (OR) neurons in the olfactory epithelium of the
nose. About 100 genes that encode ORs have been identified in zebrafish (Kratz et al. 2002; references therein).
Four main classes of water-soluble odorants are detectable
by the fish olfactory system: amino acids, gonadal steroids,
bile acids and prostaglandins (Laberge & Hara 2001).
Electrophysiological and activity-dependent labeling techniques have been employed to investigate the capability of
zebrafish to detect different odorants. Recently, polyamines
as well as the monoamine histamine have been shown to
elicit significant olfactory responses, and it was found that a
novel transduction pathway that is distinct from the ones
used by amino acids or bile acids may mediate the response

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Genes, circuits and behavior in zebrafish

to polyamines (Michel et al. 2003). In addition, optical imaging of neuronal activity in the olfactory bulb has been
employed to determine the structural requirement and minimal number of different zebrafish olfactory receptors that
respond to a series of naturally occurring amino acids and
structurally related compounds. This analysis suggests that
odorant detection requires several different receptors even
for relatively simple odorants, and individual receptors
require the presence of certain molecular features represented in the odorant molecules (Fuss & Korsching 2001).
Based on the effects on animal behavior, odorants can also
be classified as chemo-attractants, repellents or neutral stimulants. Zebrafish are attracted to amino acids and repelled by
copper (Steele et al. 1990). How different odorants elicit
attractive or aversive behavior in vertebrates is likely to be
complex and involve not only the sensory system but also
neuromodulatory as well as motor systems. Saturating
genetic screens of chemoattractive/chemorepellent behavior
has been carried out in C. elegans (Bargmann et al. 1993),
which identified highly conserved odorant receptor genes and
elucidated sensory neuron axon guidance. However, genes
and pathways that are involved in mediating the attraction/
avoidance behaviors have not been identified in C. elegans. In
addition, the simple C. elegans nervous system is quite different from the vertebrate olfactory system, which contains the
olfactory epithelium, the bulb and higher processing centers.
Therefore, future genetic study of chemoattractive or chemorepellent behavior in zebrafish may provide important insights
into the understanding of molecules and neural network in
mediating such attraction/avoidance behavior.

Hearing and mechanosensation

The vertebrate inner ear is the organ of hearing and balance.
Defects within the neuroepithelium of the inner ear can lead
to congenital deafness in humans. Although the zebrafish ear
does not contain the specialized hearing organ cochlea, other
features are preserved in zebrafish. Specialized sensory hair
cells are found within the zebrafish inner ear. In addition, hair
cells are also present in the lateral line neuromasts that are
involved in balancing and detecting water flow. A genetic
analysis of inner ear development and function will provide
insights into understanding human deafness as well as
mechanisms underlying mechanosensory transduction.
Since the zebrafish ear is readily visible in developing
embryos, simple screens for defects in ear morphology
have been carried out (Malicki et al. 1996b; Whitfield et al.
1996). In addition, behavioral assays were also employed:
since the inner ear functions in maintaining balance, mutations that show defects in balance were isolated (Granato
et al. 1996). The circler type of zebrafish mutants have
trouble keeping their postural balance, often swim on their
sides or back, and many fail to exhibit the acoustic/vibrational
startle reflex. The acoustic/vibrational startle reflex is present
in 72-h-old larvae and involves a relatively simple neural
circuit: the sensory hair cells activated by auditory or vibraGenes, Brain and Behavior (2004) 3: 6374

tional stimuli cause the firing of the eigth cranial sensory

ganglia, which activate the Mauthner neurons. The Mauthner
neurons subsequently activate motor neurons that output
the startle response. Monitoring the activity of these
neurons through calcium imaging analysis revealed that in
many of the circler mutants these neurons do not display
calcium signals in response to vibration but do so in response
to touch. This analysis suggests that these circler mutants
have defects in the periphery involved in sensing vibration,
whereas the hindbrain neurons are likely to be normal. Gross
and fine structure analysis of inner ear as well as recordings
from the lateral line hair cells allowed further classification of
circler mutants into five different classes (Nicolson et al.
1998). Among the circler mutants, the molecular identity of
mariner has been revealed to encode myosin VIIA (Ernest
et al. 2000). Mutations in this gene are also linked to human
deafness, the Usher 1B syndrome, thus validating zebrafish
as a model for human hearing disorders. Recently, a morpholino-based reverse genetic analysis of a zebrafish homologue of the Drosophila NompC TRP channel revealed that it
plays an essential role in vertebrate sensory hair cell mechanotransduction (Sidi et al. 2003).
In addition to screening for balancing mutants, a dominant
screen for hearing defects has been conducted (Bang et al.
2002). The assay monitors a rapid escape reflex in response
to a loud sound. Over 6500 fish were tested, and 1% of them
were found to be non-responsive. Subsequent X-ray imaging
analysis revealed defects in conductive elements of the auditory system. However, none of the putative mutations were
transmitted to the next generation, suggesting that these mutations were somatic rather than germ-line in nature. It is possible
that a loss of two copies of a particular gene is necessary to
produce a heritable hearing mutant phenotype; therefore, a
recessive screen using the assay may turn out to be fruitful.

The motor system

The first motor behavior of zebrafish is a spontaneous contraction at 17 h after fertilization, and it depends upon a
simple spinal network consisting of fewer than 20 primary
motor-, sensory- and interneurons (Saint-Amant & Drapeau
1998; Saint-Amant & Drapeau 2001). By 27 h old, embryos
respond to a gentle touch by swimming away for a short
distance. More frequent spontaneous beat-and-glide swimming begins at 5 days. In addition, 5-day-old larvae acquire
sensory cue-induced reflective movement such as the optomotor response (visual cue) and escape behavior (mechanosensory cue), as well as goal-directed motion (e.g. feeding,
and escape from predators).
Calcium imaging in combination with laser lesioning has
been employed to study neuronal circuits regulating larval
escape in response to tactile stimuli (Liu & Fetcho 1999;
OMalley et al. 1996). Calcium indicators loaded into hindbrain neurons through back filling allowed the monitoring of
the activity of labeled neurons in living zebrafish. Further-

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more, specific laser killing of selected reticulospinal neurons

leads to delayed behavioral response, functionally validating
the role of these neurons in the behavior.
A mutant named space cadet was isolated for its locomotion defect (Lorent et al. 2001): normal larvae exhibit a
C-bend pattern of escape upon tactile or vibrational stimuli,
followed by accelerated swimming through a series of fast,
bilateral tail flextures. However, the space cadet mutant
often performs multiple C-bend towards the same side,
resulting in circumferential rotation. In addition, the mutant
displays repeated tail flextures to the same side. Detailed
cellular study revealed that the motor defect is correlated
with axonal defects in a small population of commissural
hindbrain spiral fiber neurons. Severing their axons in the
wildtype can phenocopy the mutant phenotype, strongly
suggesting that the space cadet gene is involved in the
development of the circuitry modulating fast turning. An
additional commissural axonal defect in the retina suggests
that space cadet may have a general role in commissural
neuron axonal pathfinding.
One has to keep in mind that in addition to the neural
system, body structures or muscle strength can affect movement. The effect of fin size was recently found to affect the
swimming performance, swimming behavior and routine
activity of zebrafish (Plaut 2000). Therefore, in a genetic
screen that uses locomotion as a read-out, both neural and
non-neural mechanisms need to be taken into consideration.

coeruleus, the brain noradrenergic center. Serotonergic (5HT)

neurons are found in the basal forebrain (mainly the hypothalamus) and hindbrain (raphe nuclei). These neurons innervate
many different regions of the brain and spinal cord, and play
important modulatory roles in regulating locomotor coordination, neuro-endocrine and vital organ function, motivation and
reward, emotional balance, mood, attention, planning and
social behavior (Goldstein et al. 1998). Dysfunction of monoamine systems in humans is associated with movement
disorders, drug and alcohol addiction, anxiety and depression
and schizophrenia. In the following paragraphs I will discuss
our work on studying the effects of drugs of abuse on zebrafish and anxiety-like behavior in zebrafish, as well work from
other laboratories on several complex behaviors.

Behavior induced by drugs of abuse in zebrafish

Addictive substances (e.g. drugs of abuse, alcohol) possess
powerful reinforcing properties that lead to compulsive seeking behavior. In mammals, the brain dopamine system,
mainly the ventral tegmental area (VTA) and their projection
territory, namely the nucleus accumbens (NAC), are heavily
implicated in the process. Although a theoretical framework
exists, the molecular and cellular basis of drug-seeking is not
well understood. Can zebrafish genetics be used to dissect
the molecular components as well as cellular circuitry
involved in drug-seeking behavior?

Effects of drugs of abuse on locomotor activity

Neuromodulation by brain monoamine systems

Behaviors such as OKR, OMR and escape in response to
touch are mostly reflexive in nature, thus relatively simple
and usually involving simpler neuronal circuits. In contrast,
behaviors that are goal-directed (driven by the intention to
survive or reproduce, e.g. food hunting, escape from predators) or emotion-related (e.g. aggression, anxiety and fear)
are considerably more complex. In addition to the requirement of sensory or motor systems, neuromodulatory systems (e.g. dopamine, noradrenaline and serotonin systems)
play important roles.
Dopamine, noradrenaline and serotonin belong to the
family of monoamines (Bjorklund & Hokfelt 1984). They are
synthesized from the dietary amino acids tyrosine (for dopamine and noradrenaline) and tryptophan (for serotonin)
through a series of enzymatic reactions (Fig. 4). The key
enzymes involved in the synthesis of dopamine and noradrenaline are TH (tyrosine hydroxylase) and d.b.h. (dopamine
beta-hydroxylase). TPH (tryptophan hydroxylase) is involved
in the synthesis of serotonin. L-AADC (L-aromatic amino acid
decarboxylase) is involved in synthesizing all three monoamine neurotransmitters.
In the vertebrate central nervous system, the dopaminergic (DA) neurons are located in the basal forebrain (mainly the
hypothalamus), midbrain, olfactory bulb and retina. Noradrenergic (NA) neurons are mainly present in the locus

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Previous studies have shown that many drugs of abuse can

lead to hyperlocomotor activity when administered acutely,
and such hyperlocomotor activity may involve the same brain

Figure 4: The enzymatic pathway for dopamine,

noradrenaline, and serotonin synthesis. TH, tyrosine
hydroxylase;
L-AADC,
L-aromatic
amino
acid
decarboxylase; DBH, dopamine bete-hydroxylase; TPH,
tryptophan hydroxylase.
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Genes, circuits and behavior in zebrafish

dopamine system that mediates the rewarding effect of

drugs (Koob et al. 1998; Phillips & Shen 1996). Therefore,
one way to study the effects of drugs of abuse is to examine
the locomotor behavior upon their acute or chronic administration. Genetic screens for locomotor sensitivity to alcohol
have been carried out in Drosophila, and a mutation named
cheapdate (allelic to amnesia, a previously isolated mutant
defective in olfactory learning) was identified (Moore et al.
1998). The mutation disrupts a neuropeptide that is involved
in the activation of the cAMP pathway.
Compared to Drosophila, the nervous system of zebrafish
is more comparable to that of humans. If simple and robust
assays that assess the effects of drugs of abuse can be
developed in zebrafish, subsequent genetic study could provide important insights into the molecular and cellular
mechanisms underlying addiction. Administrations of alcohol
to adult zebrafish lead to dose-dependent modification of
locomotor activity (Gerlai et al. 2000): at low or intermediate
doses, zebrafish exhibit hyperactivity, whereas at high
doses, zebrafish become hypoactive and intoxicated. In addition to affecting locomotor activity, alcohol also modifies a
number of interesting behaviors including aggression, social
behavior, and antipredatory behavior. The effects of alcohol
are found to be strain-dependent, suggesting that genotypic
difference is accountable for alcohols effects (Dlugos &
Rabin 2003). Besides acute effects, chronic ethanol exposure was examined in Wildtype (in this case, fish with steelblue body stripes that were purchased from commercial
sources) and LFS fish (a strain with long dorsal and pectoral
fins). It was found that chronic exposure to 0.5% ethanol for
two weeks caused the wild type to swim in a less clustered
pattern, whereas LFS fish appeared more clustered. It will be
interesting to determine whether chronic ethanol exposure
can lead to tolerance, as has been observed in mammals as
well as Drosophila (Scholz et al. 2000).
Since larval zebrafish are much easier to handle in behavioral studies and genetic screens, and larval zebrafish are
particularly amenable to the study of cellular circuitry, the
response of larval zebrafish to alcohol was recently explored
(S. Bjerke, B. Lockwood, K. Kobayashi & S. Guo submitted).
Larval zebrafish are much less sensitive to handling. They are
small-sized and exhibit little social interaction; therefore, a
group of larval zebrafish can be tested in the same tank. Like
adult zebrafish, larval zebrafish exhibit acute sensitivity to
alcohol in a dose-dependent manner. Furthermore, the
response is sensitive to genetic background, suggesting that
it is genetically modifiable. Ethanol-induced hyperlocomotor
activity can be blocked by the addition of a dopamine antagonist,
suggesting the involvement of the brain dopamine system.
Similar dopaminergic regulation of ethanol-induced hyperlocomotor activity has been observed in rodents and Drosophila
(Bainton et al. 2000; Phillips & Shen 1996). In addition, it is also
observed in larval zebrafish that alcohol can modify melanosome dispersion in pigment cells. Melanosome dispersion can
be regulated through background adaptation. It is not clear
Genes, Brain and Behavior (2004) 3: 6374

whether such a response is mediated through the central nervous system, or whether it is mediated through the direct effect
of alcohol on pigment cells. Nevertheless, it provides a direct
and robust measure of the biological effects of ethanol in vivo.
The finding that larval zebrafish exhibit similar alcohol-induced
behavior as adult fish provides an easy means to explore the
genetic basis of the biological effects of alcohol.

Effects of drugs of abuse on preference behavior

Although locomotor response to drugs of abuse is a simple
way of measuring their biological effects, a more direct way
to measure motivation and reward is to examine their preference behavior. Conditioned place preference (CPP) is
a relatively simple assay for reward-related behavior
(Tzschentke 1998). In CPP, an animal learns to prefer an
initially neutral set of stimuli (conditioned stimuli, CS) that is
paired with drug exposure (unconditioned stimuli, UCS), and
the preference is assessed by the time difference the animal
spends in the drug-paired side before and after the conditioning sessions (Fig. 5). In addition to CPP, the self-administration
paradigm has been developed in rodents, in which the
rewarding effect of a drug is reflected through the animals
action of direct drug intake (Tzschentke 2001).
Using the CPP assay, cocaine directly administered in the
tank water was shown to elicit a preference response in adult
zebrafish (Darland & Dowling 2001). Only a single trial of

Figure 5: The scheme for CPP assay. A single adult zebrafish is

pre-exposed to the drug in a confined compartment, and tested
for the time spent before and after drug exposure in the
compartment.

69

Guo

visual-cue paired drug exposure is required to elicit a preference. Maximal preference (15%) was observed with 10 mg/l
cocaine concentration, whereas both lower and higher concentrations of cocaine elicited a smaller preference response.
Similarly, zebrafish were found to exhibit preference toward
morphine (S. B. Bretaud and S. G. Guo, unpublished data).

Anxiety and fear

Anxiety and fear are emotional states generated in response
to perceived or real threats, respectively. Within normal
ranges, these emotional states serve to protect individuals
from dangerous situations. However, disorders arise when
anxiety and fear are in excess and out of control. Anxiety
disorders are quite heterogeneous and many forms have
been diagnosed in humans. More than 19 million people in
the US alone are affected by one form or another. Compounds that are used to reduce anxiety in humans act on
GABA, serotonin and dopamine systems (Lister 1990).
Adequate animal models are needed to study the molecular genetic basis of anxiety and fear. In developing such
animal models, it is important to keep in mind that since
anxiety is a very complex emotion and involves many different neural systems, a particular behavioral paradigm in an
animal model is unlikely to truly mimic the complex human
disorders. Therefore, the goal of animal study shall be to
develop paradigms that assess anxiety-like behavior in animals and study the molecular and cellular mechanisms in the
context of the specific animal behavior. Genes and circuitry
identified in animal study will provide candidates for subsequent investigation in humans.
Over the years, several behavioral paradigms have been
established in rodents that are likely to measure the state of
anxiety or fear in the tested animal, as drugs that are known
to reduce human anxiety can also modify such behavior in
animals (Finn et al. 2003; Lister 1990). Some of these behavioral paradigms test exploratory behavior (open field, the
plus-maze test and light-dark transition), as animals tend to
explore less when in a state of anxiety or fear. Other paradigms also measure social behavior, as anxiety or fear
decreases social interaction.
Given the strength of zebrafish for forward genetic studies, can it be used to dissect the molecular and cellular
basis of anxiety-like behavior? Recent studies have started
to address this question. It has been previously noted that
adult zebrafish show a natural preference for the dark environment when tested in a tank that is half-white and halfblack (Serra et al. 1999). A quantitative test showed that both
adult and larval zebrafish exhibit 80% preference for the
dark side in a light/dark preference chamber. Furthermore,
anxiolytic compounds can modify such behavior (B. L. Lau
and S. G. Guo, unpublished data).

Other complex behaviors of zebrafish

Cereberal lateralization, once thought to be a human-specific
feature, has been found to be widespread in vertebrates

70

(Vallortigara et al. 1999). Behavioral lateralization is revealed

in zebrafish by preferential eye use (Miklosi & Andrew 1999;
Miklosi et al. 1998; Miklosi et al. 2001). The way zebrafish
view a range of different objects and react to these objects
was behaviorally recorded. It was found that the right eye
was used at the first encounter of an object (e.g. a colored
bead), during which a decision has to be made regarding
what action to take (e.g. to bite or not to bite). Subsequently,
when the object becomes familiar due to habituation, the left
eye was used. These findings have several implications.
First, in zebrafish, the left and right hemifield carry out different tasks: the left hemifield may have a role in inhibiting a
reaction before a decision can be reached. Second, this
behavioral paradigm also serves as a way to measure decision-making and goal-directedness. It will thus be interesting
to see if monoamine systems play a role in mediating such
behavior.
A simple spatial alternation paradigm with a food reward
was recently employed to assess learning and memory in
zebrafish (Williams et al. 2002). Fish were fed on alternating
sides of a divided fish tank, with a red card displayed on one
side to serve as a visual cue for orientation. Observations
were made at three time points (at the cue, a light tap near
the center of the tank, during food delivery, and 5 seconds
after the food delivery), and correct responses were compiled for averages and statistical purposes. It was found that
adult zebrafish quickly learned to alternate for food, and
could recall the learned task after a short period of 10 days.
The alternating behavior was extinguished by withholding
the food reward. Furthermore, 68-week-old juveniles
learned the task as well as or better than adult fish, whereas
34-week-old zebrafish did not learn the task to any significant level above random chance. It is possible that 34week-old fish are too young to learn or they are too weak
to properly complete the task. Taken together, this analysis
suggests that higher mental processes such as learning and
memory are present in zebrafish, and may be subsequently
studied at molecular and cellular levels, harnessing the
power of zebrafish forward genetics. It will be interesting
to test pharmacologically what neurotransmitter systems
(e.g. the monoamine system) may be involved in modulating
this behavior.

A genetic analysis of the development of the

monoamine system
The developmental ontogeny of DA, NA and 5HT neurons in
zebrafish has been characterized (Bellipanni et al. 2002; Guo
et al. 1999b; Ma 2003; Rink & Wullimann 2001). Similar to all
teleosts examined to date, the majority of DA neurons are
found in the basal forebrain while absent from the midbrain.
Anatomical as well as tracing experiments have shown that
the teleost basal forebrain DA neurons send ascending projections to telencephalon, particularly to the proposed area of
striatum (Parent et al. 1984; Rink & Wullimann 2001). Thus,
the telesot basal forebrain DA neurons may carry out funcGenes, Brain and Behavior (2004) 3: 6374

Genes, circuits and behavior in zebrafish

tions similar to the mammalian midbrain DA neurons. In

addition, DA neurons are found in the olfactory bulb and
retina. NA neurons are present in very small numbers (310
in adult zebrafish) in the locus coeruleus. 5HT neurons are
found in the epiphysis, basal diencephalon and hindbrain.
Because DA neurons selectively degenerate in Parkinsons
disease patients, elucidating the mechanisms of DA neuron
development will provide important insights into the regeneration of these neurons. A genetic screen for mutations
affecting the development of DA and NA neurons has identified a handful of mutations (Guo et al. 1999b). The foggy
mutant exhibited a deficit of DA neurons but a surplus of 5HT
neurons in the basal diencephalon, and was found to disrupt
a regulator of transcription elongation (Guo et al. 2000). This
is the first example to show that regulation of transcription
elongation plays an important role in vertebrate neuronal
development. However, the foggy mutant also has defects in
other neuronal types, e.g. neurons in the retina. This precludes
its use in addressing the function of basal forebrain DA and
5HT neurons. Nevertheless, the foggy mutant opens a window
to look into the role of transcription elongation in regulating
neuronal fate determination.
The soulless mutant lacks the locus coeruleus NA neurons.
These neurons send their projections widely throughout the
entire brain and regulate a variety of functions, such as
mood, alertness, sleep-wake and memory acquisition
(Barnes & Pompeiano 1991; Cirelli & Tononi 2000). Like DA
neurons, locus coerulus NA neurons are also vulnerable in
neurodegenerative disorders such as Parkinsons disease
and Alzheimers disease. The soulless gene encodes the
paired homeodomain transcription regulator phox2a that is
required to specify the locus coeruleus noradrenergic
neurons together with subsets of cranial sensory and motor
neurons (Guo et al. 1999a). The soulless mutant is larval
lethal, due to apparent lack of food intake. The exact
neuronal cause of this phenotype is not yet clear.
The too few mutant, in which the basal forebrain DA and
5HT neurons are selectively reduced, disrupted a zinc-finger
containing protein that probably acts as a transcription regulator
(Levkowitz et al. 2003). Among the mutants isolated, the too
few mutant showed the most specific defects in that only DA
and 5HT neurons located in the basal forebrain were affected.
The too few mutant is adult viable, and has no observable
phenotype in terms of sensory modality or movement.
Therefore, the too few mutant provides a great opportunity
to explore the function of basal forebrain DA and/or 5HT
neurons.

the function of neural systems in living organisms. However,

behaviors that involve the monoamine system are generally
quite complex. To apply a forward behavioral genetic screening strategy to identify genes and pathways in the formation
and function of monoamine systems and possibly other
related neural systems, the challenge rests on the establishment of appropriate behavioral assays. In order for the
assays to work for a large-scale genetic screen, they must
have the features of simplicity and robustness. Since it is not
possible for a single behavioral assay in any animal model to
truly resemble complex human disorders, the approach shall
be to understand the behavior at molecular and cellular levels
in the context of a particular model organism. Genes and
circuitry identified in animal study will provide much-needed
candidates for subsequent investigation in humans.
A pilot screen has been carried out to identify mutations
that showed altered CPP response to cocaine (Darland &
Dowling 2001). Three mutations that displayed abnormally
low response to cocaine were identified. Greater than 45%
of individuals in the F2 family from all three mutations
showed insensitivity to cocaine, suggesting that all three
mutations are dominant. Although the frequency of recovering mutations appears to be overly high (3 out of 18 families
screened), the behavior was inherited by the F3 generation in
a manner that suggests they are dominant single gene mutations. Given this frequency, one would expect that there are
many genes that are involved in cocaine CPP and can be
mutated to give an identifiable phenotype.
Other behavioral assays described above (e.g. the ethanolinduced locomotor activity, or the light/dark preference) are
relatively simple for high throughput genetic screens. The
assays that measure behavioral lateralization and learning
and memory are very interesting. However, more work is
needed to amend them to a higher throughput. It will
be interesting to test the too few mutant in these assays
and subsequently use these assays for forward genetic
screens. Sensory or motor defective mutants will affect
these behaviors, but they can be easily distinguished by
behavioral assays that assess sensory or motor integrity.
Mutants identified could be involved in the development,
neural connectivity and function of monoamine systems as
well as other neural systems involved in these complex
behaviors such as motivation and reward, anxiety/fear, decision-making, functional lateralization and learning and
memory.

A genetic analysis of the function of the monoamine

system

The past three decades have seen zebrafish grow from a pet
store resident to a laboratory model organism. With the
build-up of genetic and genomic infrastructure, the development of behavioral assays and the strength for large-scale
forward genetic screens, the zebrafish is expected to make
valuable contributions to the understanding of genes, brain,
behavior and human neurological disorders.

The monoamine systems carry out a wide variety of functions, including motor coordination, motivation and reward,
anxiety/fear, social interaction and learning and memory, and
their dysfunctions are implicated in a range of complex
human disorders. A behavioral approach is desirable to reveal
Genes, Brain and Behavior (2004) 3: 6374

Concluding remarks

71

Guo

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Acknowledgments
The author wishes to thank Brent Lockwood, Keerthi Krishnan
and an anonymous reviewer for helpful comments on the manuscript. Work in the authors laboratory is supported by grants
from Searle Scholars program, Burroughs Wellcome Fund,
David and Lucile Packard Foundation, Sandler family Foundation
and NIH.

Genes, Brain and Behavior (2004) 3: 6374