meeting report - 7th international zebrafish behavioral neuroscience and neurophenotyping workshop...
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Meeting report published in STRESS, BRAIN AND BEHAVIOR journal: (www.stressbrainbehavior.com) of 7th International Zebrafish Behavioral Neuroscienceand Neurophenotyping Workshop (ZB2N), Washington DC, November 2014. This Workshop was organized by International Stress and Behavior Society.TRANSCRIPT
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STRESS, BRAIN AND BEHAVIOR*
Vol. 3, 2015, in press early
Journal homepage: www.stressbrainbehavior.com
Meeting report
7th International Zebrafish Behavioral Neuroscience
and Neurophenotyping Workshop (ZB2N),
Washington DC, November 2014
Adam D. Collier1
1 Department of Psychology, University of Southern Mississippi, Hattiesburg, MS, USA
Abstract Washington, DC (USA) hosted the 7th International Zebrafish Behavioral Neuroscience and Neurophenotyping Workshop ZB2N on November 14th and 20th, 2014. The overall aim of the one-day workshop, organized by the International Stress and Behavior Society (ISBS), was to provide a timely synopsis of the translational momentum and utility of the zebrafish model, discuss neurobehavioral applications and neurophenotyping techniques, and allow participants to present their data, discuss research problems and obtain expert consultation. An international delegation of scientists from academia, industry, as well as post-docs, graduate and undergraduate students, participated in the workshop.
Keywords: zebrafish, behavior, neurophenotyping, CNS models
Article history: Received 14 March 2015 Received in revised form 25 March 2015 Accepted 27 March 2015
The 7th International Zebrafish Behavioral
Neuroscience and Neurophenotyping Workshop (ZB2N)
was held in Washington, DC, USA, on November 14th and
20th, 2014. Hosted by the Georgetown University Institute
for Continuing Studies in downtown DC, the workshop was
organized by the International Stress and Behavior Society
(ISBS), the Zebrafish NeuroPhenome Project (ZNP) and
the International Zebrafish Neuroscience Research
Consortium (ZNRC). The one-day workshop was held in
conjunction with the Society for Neuroscience SfN2014
annual convention, and was officially sponsored by the TSE
Systems GMBH (Germany).
The ZB2N workshop consisted of presentations
covering major neurobehavioral domains and phenotyping
techniques for probing normal and abnormal behaviors in
the zebrafish (Danio rerio) model organism. It also
emphasized the utility of this aquatic species in translational
neuroscience, biomedical and biological psychiatry
research.
Professor A. V. Kalueff (USA), ISBS President and
director of the ZENEREI Institute, welcomed participants on
*Official Journal of the International Stress and Behavior Society (ISBS), ISSN 2374-3565 Open Access paper (available freely at www.stressbrainbehavior.com) Corresponding author: Adam D. Collier. Address: Department of Psychology, The University of Southern Mississippi, 118 College Drive, Box 5025, Hattiesburg, MS 49406, USA Phone:+ 1 207 318 3300; E-mail: [email protected] The International Stress and Behavior Society (ISBS, www.stressandbehavior.com)
behalf of ISBS and ZNRC. He opened the workshop by
providing an overview of the benefits of larval and adult
zebrafish models for CNS research. It was noted that
zebrafish offer a number of practical advantages, such as
being a lower vertebrate species that is transparent at early
stages, develops rapidly, reproduces abundantly, is low-
cost, and offers a unique balance between behavioral
complexity and experimental throughput. He also
emphasized that zebrafish have a high degree of genetic
and physiological homology to humans, similar central
nervous system (CNS) morphology, and possess all major
neurotransmitters, hormones and receptors. The speaker
then highlighted the evolutionarily conserved nature of
pathways and behaviors between mammals and zebrafish
implicated in the pathogenesis of brain disorders, such as
anxiety. For example, when exposed to stimuli that evoke
fear or anxiety (e.g., predator fish, alarm pheromone, or
anxiogenic pharmacological substances), zebrafish display
robust behavioral phenotypes such as reduced exploration,
scototaxis (dark preference), geotaxis (diving/bottom
preference), thigmotaxis (peripheral preference), immobility
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and erratic movements. These behaviors strongly correlate
with physiological biomarkers such as cortisol and brain c-
fos expression. The presenter noted the striking parallels in
human, rodent and zebrafish anxiety-related phenotypes,
and the value provided by such comparative approaches to
understanding and treating neuropsychiatric disorders. He
then briefly outlined several other CNS disorders zebrafish
may be used to investigate, including autism spectrum,
attention deficit/hyperactivity disorder (ADHD), post-
traumatic stress disorder (PTSD), depression, drug abuse,
and various neurotoxidromes. The speaker also provided a
description of advanced neurophenotyping techniques
such as 3D temporal and spatial reconstructions of
zebrafish swim traces. The utility of zebrafish in biological
psychiatry for drug discovery research and to investigate
molecular mechanisms underlying brain disorders was also
discussed, specifically in relation to recent genetic and
technological advancements for in vivo examinations, high-
throughput screening, and whole brain analyses of larval
and adult zebrafish.
The session continued with an informative keynote
lecture by Professor D. Echevarria (USA, Fig. 1), ISBS
Fellow, from the University of Southern Mississippi (USM),
who spoke to workshop participants about his experience
transitioning from rodent to zebrafish models and the
striking neurobehavioral parallels he has observed. Dr.
Echevarria discussed the development of a behavioral test
battery to assess global drug effects using zebrafish, which
included data on the effects of the glutamate/NMDA
receptor antagonist MK-801 (dizocilpine) in zebrafish. MK-
801 was found to decrease social behavior (shoaling) and
increase circling behavior. It was said that this behavioral
profile is consistently reported in rodents, which is
supportive of face and construct validity in zebrafish models.
The presenter also discussed the importance of observing
individual differences in behavioral research, which he
described as being more significant than just noise around
an average. He presented data on the consistency of four
zebrafish behavioral traits (i.e., aggression, boldness, fear,
and exploration) across experimental context (i.e., mirror
exposure, novel object, predator exposure, and open-field
tests). It was reported that individual differences in
behavioral traits remained stable across context and time.
The speaker concluded by noting that the identification of
individual differences in zebrafish will allow for future
investigation into underlying gene x environment
interactions.
Dr. H Russig (Germany, Fig. 1), Scientific Director of
TSE Systems, started the afternoon session by focusing on
recent developments in fully automated behavioral test
equipment for zebrafish research. One such system,
recently developed by TSE Systems, is VisioTrainer, in
which the cognitive performance and visual system
properties of up to eight zebrafish may be simultaneously
evaluated within visual discrimination tasks. For example,
zebrafish may be placed in a neutral start zone and allowed
access into two choice zones in which arbitrary visual
stimuli are presented on LCD screens, as defined by the
experimenter. As zebrafish enter the predetermined correct
choice zone they are rewarded with food. The speaker
presented data on the improvement in stimulus
discrimination performance over time, the extinction of this
learned response when the stimuli were removed, and the
occurrence of reversal learning when the correct choice
was reversed. Multiple behavioral parameters may be
automatically tracked and analyzed throughout the testing
period. The second automated system Dr. Russig
presented was VisioTracker, which tracks and quantifies
visual oculomotor performance in larval or adult zebrafish
via the highly conserved optokinetic response (OKR).
Valuable tools such as the aforementioned were described
to reduce personnel time, eliminate observer bias, and
permit researchers to accurately and exhaustively dissect
zebrafish phenotypes.
In his talk, Professor J. Kunicki (Poland), Director of Warsaw-based International Institute of Molecular and Cell
Biology (IIMCB), introduced the strategic goals of the
recently initiated EU-funded FishMed project. This project
capitalizes on the advantageous characteristics of the
zebrafish model to investigate molecular mechanisms and
targets for therapeutic intervention of diseases such as
cancer, Alzheimers and Parkinsons. He also described the
Be Healthy as a Fish educational campaign, which
includes free workshops designed to introduce 5th and 6th
grade elementary school students to zebrafish research
and describe how this field improves human health. Such
informal educational workshops also allow students to take
part in discussions, perform biological experiments and
observe zebrafish under a microscope.
The remaining workshop presentations were given by
junior researchers from The University of Alberta (UA),
Stony Brook University (SBU) and the University of
Southern Mississippi (USM). A. Collier (USM) first provided
a review of zebrafish models of drug abuse, noting that
zebrafish demonstrate robust behavioral and physiological
phenotypes related to drug abuse and addiction that are
markedly comparable to rodent and clinical findings,
specifically with regard to drug sensitivity, tolerance,
withdrawal, relapse, endocrine effects, cognitive deficits,
and drug reward. He then presented current data on the
rewarding effects of alcohol, nicotine and caffeine in adult
zebrafish in the conditioned place preference (CPP)
paradigm. During CPP testing, zebrafish were first allowed
to explore an apparatus divided into two visually
discriminative environments (dots vs. no dots), and the
initial preference to spend time in each environment was
measured. On the following day, zebrafish were then
conditioned with a dosage of drug via immersion in their
least preferred side over a number of conditioning sessions.
During the final day of testing, zebrafish were once again
allowed to explore the entire apparatus and were probed
once again for their preference to spend time in each
environment. Notably, ethanol and nicotine, but not caffeine,
produce robust CPP behavior in zebrafish following a single
conditioning session in a dose-dependent manner. The
presenter concluded by noting that the evolutionarily
conserved nature of drug abuse behavioral phenotypes and
the functional homology of pathways (e.g., dopamine) in the
zebrafish brain, in addition to shared genes related to
addiction in humans, establish the zebrafish as a powerful
in vivo model of drug abuse.
C. Moravec (SBU, USA) started the evening sessions
presentations by discussing zebrafish models of neural
development. C. Moravec described the role of the
maternal RE1-silencing transcription factor (Rest) in
regulating the expression of neural specific genes, and how
little is known about the requirement for Rest in behavior,
partly due to embryonic lethality in the mouse knockout.
Their laboratory crossed zebrafish rest mutant females with
wild type males, creating a zebrafish line (Mrest) that lacked
maternal rest but had zygotic rest. Mrest mutant larvae
were found to exhibit hyperactivity and increased
thigmotaxis, but had normal startle responses to light and
acoustic stimuli. At 6 months of age, Mrest adult males, but
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not females, displayed increased thigmotaxis in a novel
environment. Rest mutant larvae, which had maternal rest
but not zygotic rest, displayed hypoactivity and no increase
in thigmotaxis. She noted that these results help establish
a link between Rest and its function in controlling behavior,
in that maternal Rest was found to modulate larval and adult
zebrafish neurophenotypes in a sex specific manner. The
speaker concluded by describing the zebrafish as a
powerful model in developmental neuroscience, chiefly due
to the genetic tractability, high degree of homology, and
robust behavioral repertoire of this species.
Figure 1. The Workshop agenda (left to right, top down): faculty and participants; talks by Prof. D. Echevarria (USA) and Dr.
H. Russig (Germany); Prof. A. Kalueff presenting Ms F. De Santis (France) a travel award on behalf of ISBS and ZNRC.
P. Leighton (UA, Canada) presented her research on
zebrafish models of Alzheimers disease, in which she
employed targeted mutagenesis to investigate the roles of
prion protein (PrP) and amyloid precursor protein (APP). It
was noted that zebrafish possess functionally conserved
homologs of PrP (prp1 and prp2) and APP (appa and appb).
Their laboratory created a zebrafish mutant via zinc finger
nucleases with a null prp2 allele, exposed mutagenized
larvae to the convulsant pentylenetetrazole (PTZ), and
used in situ hybridization and qPCR to detect c-fos
expression. Interestingly, mutant larvae were found to have
increased c-fos expression following PTZ treatment and
greater sensitivity to PTZ-induced seizures than wild type
larvae, a comparable phenotype to that observed in PrP
knockout mice. Preliminary data was presented, showing
that prp2 mRNA can rescue this phenotype but wildtype
mRNA cannot. P. Leighton also described the use of
transcription activator-like effector nucleases (TALEN) to
specifically target zebrafish appa and prp1 and the creation
of mutant fish to be used to investigate interactions
between PrP and APP. P. Leighton noted that an
understanding of the function and relationship of such
genes will generate knowledge that will help to identify
therapeutic targets for prion and Alzheimers disease in humans.
E. Caramillo (USM) presented her research on
learning acquisition in zebrafish within the T-maze test and
the effects of methylene blue exposure on cognitive
performance. E. Caramillo began by discussing the
capacity of zebrafish to exhibit both associative and non-
associative forms of learning, such as classical and operant
conditioning, as well as habituation responses. The
methodology of the zebrafish T-maze test was then
described. First, a single zebrafish was allowed to choose
to enter one of two arms differing in stimulus properties and
were rewarded with food if the predetermined correct
choice was made. A correction trial took place following an
incorrect choice, in which fish were only allowed to enter
the correct arm. This training procedure took place 4 times
a day for 5 days. Immediately following the final training
session experimental animals were exposed to a dosage of
methylene blue for 12 hours and then returned to their
home tanks for another 12 hours. Zebrafish were then
probed for learning acquisition on the final day of testing.
Recent data was reported on improved cognitive
performance during the probe trial in zebrafish treated with
methylene blue. She concluded her presentation by noting
that cognitive impairment is a symptom of several
neuropsychiatric disorders such as Alzheimers disease and schizophrenia, and that zebrafish models may aid in
the discovery of novel cognitive enhancers with therapeutic
potential.
K. Khan (USM) concluded the Program with a
discussion on zebrafish sleep models and recent
preliminary data on the anxiogenic effects of sleep
disruption. She evaluated sleep-wake behaviors and
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anxiety profiles of groups of 3 fish that were housed under
either continuous light for 24 hours, continuous darkness
for 24 hours, or under normal laboratory lighting conditions
of 14 hours of light and 10 hours of darkness. K. Khan
reported that 24 hours of light prevented sleep from
occurring at various time points and increased anxiety-like
behaviors in the novel tank test. The presenter described
zebrafish as a useful sleep model due to highly conserved
mechanisms controlling sleep and wake behavior. For
example, like humans, zebrafish are a diurnal species with
circadian rhythms mediated by light input and possess a
hypocretin/orexin system that promotes wakefulness. She
noted that, in humans, acute and chronic sleep deprivation
may result in negative outcomes such as cognitive deficits,
weight gain, immune dysfunction and the development of
affective disorders, and that zebrafish may provide
significant insight into processes associated with the sleep
disorder pathogenesis.
The popularity of zebrafish in neuroscience research
has grown markedly in recent years. Laboratories are
increasingly recognizing that zebrafish are a powerful
model organism that exhibit robust behaviors, have a high
degree of genetic, neural and physiological homology to
mammals, and offer numerous practical advantages, such
as low-cost, ease of handling, high fecundity, and rapid
development. Zebrafish models are emerging throughout
various domains of research, and such a burgeoning field
is undoubtedly progressed by workshops such as ZB2N.
The workshop provided a cordial atmosphere with
ample time for interdisciplinary dialogue and informal group
discussions. A travel award to a young international
scientist (to Ms F. De Santis, U Marie Curie, France) was
presented by ISBS on behalf of the Visitor Guard Insurance
Company (USA, www.visitorguard.com). The workshop
abstracts were published in Stress, Brain and Behavior (this
Issue). Concluding the workshop, it was announced that the
next International ISBS/ZNRC zebrafish workshops
(www.stressandbehavior.com) will be held in Miami, FL on
June 24, 2015 (in conjunction with the 5th ISBS North
American Stress and Behavior meeting), and on October 16/24 in Chicago, IL (in conjunction with the SfN2015
annual meeting).
Conflict of interest
The author declares no conflicts of interest