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: adam.d.collier@eagles.usm.edu 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