Behavioral and Neural Genetics of Zebrafish

Behavioral and Neural Genetics of Zebrafish assembles the state-of-the-art methodologies and current concepts pertinent to their neurobehavioral genetics. Discussing their natural behavior, motor function, learning and memory, this book focuses on the fry and adult zebrafish, featuring a comprehensive account of modern genetic and neural methods adapted to, or specifically developed for, Danio rerio. Numerous examples of how these behavioral methods may be utilized for disease models using the zebrafish are presented, as is a section on bioinformatics and "big-data" related questions.

• Provides the most comprehensive snapshot of the fast-evolving zebrafish neurobehavior genetics field
• Describes behavioral, genetic and neural methods and concepts for use in adult and larval zebrafish
• Features examples of zebrafish models of human central nervous system disorders
• Discusses bioinformatics questions pertinent to zebrafish neurobehavioral genetics

• Language: English
• Publisher: Academic Press
• Release date: Apr 3, 2020
• ISBN: 9780128175293

Book preview

Behavioral and Neural Genetics of Zebrafish

Editor

Robert T. Gerlai

Department of Psychology, University of Toronto, Mississauga, ON, Canada

Table of Contents

Cover image

Title page

Copyright

In Memoriam

Contributors

Acknowledgments

Introduction: The Zebrafish in behavioral and neural genetics

Section I. Introduction to Zebrafish: Natural habitat, ethology, and appropriate maintenance conditions

Chapter 1. The biotic and abiotic environment of zebrafish

Introduction

Geographic distribution and phylogeny

Features of the abiotic and biotic environment

Environmental changes to zebrafish habitat

Conclusions

Chapter 2. Maintenance and breeding of zebrafish, with some ethological and ecological considerations in mind

Introduction

The question of standardization

Zebrafish in nature

Laboratory maintenance practices

Absolute value versus speed of change of water parameters

Conditions in the experimental versus holding tanks

Concluding remarks

Section II. The behavior of the fry

Chapter 3. The larval visual system and behavioral responses to visual stimuli

Relevance of vision for the zebrafish larva

General structure of the zebrafish visual system

Retinal projections

Visual processing

Comparison of visual processing in zebrafish and mammals

Visual behavior

Chapter 4. Visual escape in larval zebrafish: stimuli, circuits, and behavior

Introduction

The essential properties of loom stimuli (what causes an animal to escape?)

Execution of escape behaviors

Core visual escape circuitry

Modulation of the core escape circuit

Open questions about the loom circuit

Chapter 5. Functional review of hearing in zebrafish

The underwater auditory environment and its relevance to sound detection

Morphology of the zebrafish ear

A note on the role of the ear versus the lateral line neuromasts

Meta-analysis of increasing importance of zebrafish in auditory studies

Zebrafish hearing and how we know

Hair cell dynamics

Causes of hair cell death in fish

Hair cell regeneration

Formation and growth of zebrafish otoliths

Transduction of acoustic stimulation in the zebrafish

Chapter 6. The development of social behavior

Social development across species

Defining social behavior in zebrafish

Classifying developmental stages

Onset of specific social behaviors

Social attraction

Shoaling and social preference

Social cueing

Early experience

Conclusions

Chapter 7. Classical and operant conditioning in larval zebrafish

Introduction

Classical conditioning

Classical conditioning with restrained zebrafish larvae

Classical conditioning with freely swimming zebrafish larvae

Neurobiological mechanisms of classical conditioning in larval zebrafish

Operant conditioning

Operant conditioning with freely swimming zebrafish larvae

Operant conditioning with restrained zebrafish larvae

Neurobiological mechanisms of operant conditioning in larval zebrafish

Associative learning with appetitive stimuli

General discussion

Future directions

Section III. The behavior of the adult

Chapter 8. Motor patterns and swim path characteristics: the ethogram of zebrafish

Introduction

Phenotyping of zebrafish locomotor behavior

Motor patterns of zebrafish

Motor behavior in adult zebrafish

Genetic control and locomotor mutants

Concluding remarks

Chapter 9. Behavioral criteria and techniques to define sleep in zebrafish

Introduction

Behavioral criteria to characterize sleep

Circadian regulation of the sleep/wake cycle

Sleep deprivation and homeostasis

Arousal threshold

Sleeping posture

Future tools and criteria to define sleep

Chapter 10. Fear responses and antipredatory behavior of zebrafish: a translational perspective

Introduction

Anxiety or fear?

Fear: a natural response to predators

Visual stimuli and their method of delivery to induce fear in adult zebrafish

Visual stimulus-specific antipredatory responses

Olfactory cues inducing antipredatory responses

Anxiety-inducing paradigms

Fear responses of the fry

Psychopharmacology of zebrafish fear and anxiety

Genetic approaches in the analysis of fear and anxiety in zebrafish

Conclusions

Chapter 11. Social behavior and its psychopharmacological and genetic analysis in zebrafish

Collective movement: schooling and shoaling

Social choice

Other paradigms

Conclusions

Chapter 12. Associative and nonassociative learning in adult zebrafish

Introduction

Nonassociative learning

Associative learning

Operant conditioning

Conclusion

Chapter 13. Relational learning in zebrafish: a model of declarative memory in humans?

Introduction

The practical advantages of zebrafish

Why study learning and memory, and why relational memory

Spatial learning, a form of relational learning

Spatial learning and memory in zebrafish versus mammalian species

Predictive and construct validity of zebrafish spatial learning?

High-throughput applications

Concluding remarks

Section IV. Genetic methods for Zebrafish

Chapter 14. Molecular genetic approaches to dissect complex behaviors in zebrafish

Introduction

The repertoire of behaviors in larval and adult zebrafish

Prepulse inhibition

Behavior screening with forward and reverse genetic strategies

Genetic stocks of zebrafish for behavioral studies

Genetic diversity across laboratory-bred and wild strains of zebrafish

The candidacy of zebrafish as a model for genome-wide association study

Transcriptomic studies for behavioral traits

Profiling transcriptomes at single cell resolution

Conclusion

Chapter 15. Behavioral research in zebrafish (Danio rerio): strain as source of variation

Introduction

Sources of variation in experimental data

Strains and genetic variation

Behavioral differences between strains

Closing remarks

Chapter 16. Designer mutants for behavioral genetics

Programmable nucleases for targeted genome modification

DNA repair and DNA modification outcomes

Target selection: potential for alternate promoters, exons, and initiation codons

Breeding practices to reduce impact of off-target or bystander mutations

Chapter 17. Optogenetics

Introduction of optogenetics

Optogenetic control of neuronal activity

Optogenetic detection of neuronal activity

Optogenetic circuit analysis

Optogenetic applications to study zebrafish behavior

Future prospects of optogenetics

Chapter 18. The CRISPR/Cas system in zebrafish

Introduction

Knock-out methodologies

Tissue-specific gene disruption

Knock-in strategies

Base editing

Transcriptional regulation

Lineage tracing

Conclusions and remarks

Chapter 19. PhOTO zebrafish and primed conversion: advancing the mechanistic view of development and disease

Introduction

A historical perspective of lineage tracing

PhOTO system and applications

Outlook

Section V. Disease models and behavior genetic applications

Chapter 20. Acute and chronic alcohol effects in zebrafish

The cost of alcohol addiction

Zebrafish: an animal model to study alcohol's effects

Acute alcohol exposure

Chronic alcohol effects

Zebrafish strain and population differences in alcohol responses

Microarrays to study chronic alcohol–induced gene expression

Reverse genetic strategies

Forward genetics and mutagenesis

Concluding remarks

Chapter 21. Behavioral genetics of embryonic ethanol exposure in zebrafish: a model for FASD

Background

Zebrafish as an animal model for FASD

Reverse genetics

Conclusion

Chapter 22. Life, death, and regeneration of zebrafish dopaminergic neurons

Development of DA neurons in zebrafish

Signaling pathways in DA neuron development

Transcription factors and DA neuron development

DA circuitry

Modeling DA neuron loss in zebrafish

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine

1-Methyl-4-phenylpyridinium ion

6-Hydroxydopamine

Rotenone

Paraquat

Chemogenetic ablation of DA neurons using nitroreductase system

Regeneration of DA neurons in zebrafish

Perspectives and challenges

Chapter 23. Parkinson's disease

Introduction

What is known about the causes of neurodegeneration in Parkinson's disease?

Role of animal models of Parkinson's disease and the potential utility of zebrafish

Suitability of zebrafish as a model for studying Parkinson's disease

Elucidating functions of genes implicated in Mendelian parkinsonism phenocopies and sporadic Parkinson's disease using zebrafish models

Elucidating mechanisms of environmental toxicants associated with Parkinson's disease, using zebrafish models

Conclusions

Chapter 24. Seizures and epilepsy

Introduction

Chemically induced seizures

Nonchemical or genetic methods to induce seizures

General aspects of epilepsy genetics

Zebrafish as a model for advanced studies in seizure and epilepsy

Chapter 25. Aging, circadian clock, and neurogenesis: the zebrafish approach

A diurnal vertebrate model

A vertebrate with gradual aging

Accelerated aging due to metabolic challenge in zebrafish

Adult neurogenesis in zebrafish is under circadian control

Neurogenesis in normal and accelerated aging

Conclusion

Chapter 26. Modeling autism spectrum disorders in zebrafish

Autism spectrum disorders—clinical features and research challenges

Advances in the genetics of autism spectrum disorders

Conserved ASD-relevant neural pathways in zebrafish

ASD-relevant zebrafish behaviors

Zebrafish ASD risk gene models—generation and phenotyping

Zebrafish modeling of ASD—current and future directions

Conclusion

Chapter 27. Studying aggression in zebrafish

Aggression and agonistic behavior

Studying aggression in zebrafish

Agonistic behavior of zebrafish during dyadic fights

Methods to quantify agonistic behavior

The development of dominance hierarchies—social stress and agonistic behavior

The neural basis of aggression

The genetic basis of aggression

The pharmacological basis of aggression

Conclusion

Chapter 28. Methylmercury induces transgenerationally transmissible epigenetic changes influencing zebrafish behavior

Introduction

Methylmercury and behavior

Effects of developmental MeHg on zebrafish behavior

Transgenerational inheritance of dysfunction

Epigenetics

SAM, MeHg, and DNA methylation

MeHg-induced transgenerational epigenetic inheritance

Conclusions and next steps

Section VI. Big data and bioinformatics

Chapter 29. The design of behavioral screening in zebrafish

Why should we screen?

What should we screen?

Why should we use behavioral phenotypes as criteria for screening?

The conceptual question of what behavioral tests really measure

Behavioral test batteries, multiple behavioral measures, overlapping behavioral tests

Organization of test batteries: top-down versus bottom-up

Endophenotypes

How to measure behavior

Concluding remarks

Chapter 30. Software tools for behavioral phenotyping of zebrafish across the life span

Introduction

Tracking activity, heart rate, and blood and gut flow in early developmental stages of zebrafish

High-throughput screening with zebrafish larvae in well plates

Automated analysis of avoidance behavior in zebrafish larvae

Detecting specific behaviors in common tests with adult zebrafish

Detecting aggressive behavior in adult zebrafish

Quantifying shoaling behavior in adult zebrafish

Tracking adult zebrafish in 3D

Concluding remarks

Supplementary data

Chapter 31. The zebrafish genome sequencing project: bioinformatics resources

A brief history of the zebrafish reference genome sequencing project

Assessing the zebrafish genome assembly

Zebrafish genome assembly gene annotation and genome browsing

Comparative sequence analysis

Variation

Sequencing and assembling the danioninae

Chapter 32. Registered, standardized, and interactive: a review of online resources for zebrafish neuroanatomy

Introduction

Collections of zebrafish anatomy data

Resources of standardized zebrafish neuroanatomy

Conclusion and outlook

Index

Copyright

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Notices

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ISBN: 978-0-12-817528-6

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Cover image: Adult zebrafish of the AB strain kept in the Gerlai Zebrafish Facility, University of Toronto Mississauga. Photo taken by Robert T. Gerlai.

Publisher: Nikki Levy

Acquisitions Editor: Natalie Farra

Editorial Project Manager: Kristi Anderson

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Typeset by TNQ Technologies

In Memoriam

In memory of Dr. Christine Hone-Buske (February 10, 1984–May 22, 2019), the first undergraduate student, and subsequently a graduate student, in my zebrafish laboratory at the University of Toronto Mississauga.

Contributors

Rida Ansari

Department of Cell and Systems Biology, University of Toronto, Toronto, ON, Canada

Neuroscience and Mental Health Program, Hospital for Sick Children, Peter Gilgan Centre for Research and Learning, Toronto, ON, Canada

Lior Appelbaum,     The Faculty of Life Sciences and the Multidisciplinary Brain Research Center, Bar-Ilan University, Ramat Gan, Israel

W. Philip Bartel,     Department of Neurology, University of Pittsburgh, Pittsburgh, PA, United States

Carla Denise Bonan,     Laboratório de Neuroquímica e Psicofarmacologia, Escola de Ciências da Saúde e da Vida, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil

Caroline H. Brennan,     School of Biological and Chemical Sciences, Queen Mary University of London, London, United Kingdom

Edward A. Burton,     Department of Neurology, University of Pittsburgh, Pittsburgh, PA, United States

Michael J. Carvan III,     University of Wisconsin-Milwaukee, School of Freshwater Sciences, Milwaukee, WI, United States

Karl J. Clark,     Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, United States

Rosane Souza Da Silva,     Laboratório de Neuroquímica e Psicofarmacologia, Escola de Ciências da Saúde e da Vida, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil

Konstantin A. Demin

Institute of Translational Biomedicine, St. Petersburg State University, St. Petersburg, Russia

Institute of Experimental Medicine, Almazov National Medical Research Centre, Ministry of Healthcare of Russian Federation, St. Petersburg, Russia

Flavia De Santis,     ZeClinics SL, IGTP (Germans Trias i Pujol Research Institute), Barcelona, Spain

Vincenzo Di Donato,     ZeClinics SL, IGTP (Germans Trias i Pujol Research Institute), Barcelona, Spain

Elena Dreosti,     Wolfson Institute for Biomedical Research, University College London, London, United Kingdom

Marc Ekker,     Department of Biology, University of Ottawa, Ottawa, ON, Canada

Amanda Facciol,     Department of Cell and Systems Biology, University of Toronto, Mississauga, ON, Canada

Gert Flik,     Department of Animal Ecology and Physiology, Institute of Water and Wetland Research, Faculty of Science, Radboud University, Nijmegen, the Netherlands

Robert T. Gerlai,     Department of Psychology, University of Toronto Mississauga, Mississauga, ON, Canada

Marnix Gorissen,     Department of Animal Ecology and Physiology, Institute of Water and Wetland Research, Faculty of Science, Radboud University, Nijmegen, the Netherlands

Fabrizio Grieco,     Noldus Information Technology BV, Wageningen, the Netherlands

Su Guo,     Department of Bioengineering and Therapeutic Sciences, Programs in Biological Sciences, Institute of Human Genetics, University of California, San Francisco, CA, United States

Amanda A. Heeren,     Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, United States

Dennis M. Higgs,     Department of Integrative Biology, University of Windsor, Windsor, ON, Canada

Ellen J. Hoffman,     Child Study Center, Program on Neurogenetics, Yale School of Medicine, Yale University, New Haven, CT, United States

Kerstin Howe,     Wellcome Sanger Institute, Cambridge, United Kingdom

Khang Hua,     Department of Biology, University of Ottawa, Ottawa, ON, Canada

Arnim Jenett,     TEFOR Paris Saclay, CNRS UMS2010, INRA UMS1451, Universite Paris Sud, Université Paris-Saclay, Gif-sur-Yvette, France

Allan V. Kalueff

School of Pharmacy, Southwest University, Chongqing, China

Almazov National Medical Research Centre, St. Petersburg, Russia

Ural Federal University, Ekaterinburg, Russia

Granov Russian Research Center of Radiology and Surgical Technologies, St. Petersburg, Russia

Laboratory of Biological Psychiatry, Institute of Translational Biomedicine, St. Petersburg State University, St. Petersburg, Russia

Laboratory of Biopsychiatry, Scientific Research Institute of Physiology and Basic Medicine, Novosibirsk, Russia

ZENEREI Institute, Slidell, LA, United States

Konstantinos Kalyviotis,     Department of Bioengineering, Imperial College London, South Kensington Campus, London, United Kingdom

Justin W. Kenney,     Department of Biological Sciences, Wayne State University, Detroit, MI, United States

Anton M. Lakstygal

Institute of Translational Biomedicine, St. Petersburg State University, St. Petersburg, Russia

Laboratory of Preclinical Bioscreening, Granov Russian Research Center of Radiology and Surgical Technologies, Ministry of Healthcare of Russian Federation, Pesochny, Russia

Han B. Lee,     Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, United States

Emmanuel Marquez-Legorreta,     School of Biomedical Sciences, The University of Queensland, Brisbane, QLD, Australia

Noam Miller,     Departments of Psychology & Biology, Wilfrid Laurier University, Waterloo, ON, Canada

Rodsy Modhurima,     Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, United States

Stephan C.F. Neuhauss,     Department of Molecular Life Sciences, University of Zurich, Zurich, Switzerland

Lucas P.J.J. Noldus

Noldus Information Technology BV, Wageningen, the Netherlands

Department of Biophysics, Donders Institute for Brain, Cognition and Behavior, Radboud University, Nijmegen, the Netherlands

William HJ. Norton,     Department of Neuroscience, Psychology and Behaviour, University of Leicester, Leicester, United Kingdom

Periklis Pantazis,     Department of Bioengineering, Imperial College London, South Kensington Campus, London, United Kingdom

David M. Parichy,     Department of Biology and Department of Cell Biology, University of Virginia, Charlottesville, VA, United States

Marielle Piber

School of Biomedical Sciences, The University of Queensland, Brisbane, QLD, Australia

School of Medicine, Medical Sciences & Nutrition, University of Aberdeen, Aberdeen, United Kingdom

John H. Postlethwait,     Institute of Neuroscience, University of Oregon, Eugene, OR, United States

David Pritchett,     School of Biological and Chemical Sciences, Queen Mary University of London, London, United Kingdom

Hanyu Qin,     Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland

Jason Rihel,     Department of Cell and Developmental Biology, University College London, London, United Kingdom

Domino K. Schlegel

Department of Molecular Life Sciences, University of Zurich, Zurich, Switzerland

Life Science Zurich Graduate School, Ph.D. Program in Molecular Life Sciences, Zurich, Switzerland

Ethan K. Scott

School of Biomedical Sciences, The University of Queensland, Brisbane, QLD, Australia

The Queensland Brain Institute, The University of Queensland, Brisbane, QLD, Australia

Alexander Stankiewicz,     BioChron LLC, Worcester, MA, United States

S.J. Stednitz,     Institute of Neuroscience, University of Oregon, Eugene, OR, United States

Ruud A.J. Tegelenbosch,     Noldus Information Technology BV, Wageningen, the Netherlands

Javier Terriente,     ZeClinics SL, IGTP (Germans Trias i Pujol Research Institute), Barcelona, Spain

Steven Tran,     Department of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, United States

Benjamin Tsang,     Department of Psychology, University of Toronto Mississauga, Mississauga, ON, Canada

Sachiko Tsuda,     Graduate School of Science and Engineering, Saitama University, Saitama, Japan

Ruud van den Bos,     Department of Animal Ecology and Physiology, Institute of Water and Wetland Research, Faculty of Science, Radboud University, Nijmegen, the Netherlands

Victor S. Van Laar,     Department of Neurology, University of Pittsburgh, Pittsburgh, PA, United States

Monica Ryff Moreira Roca Vianna,     Laboratório de Biologia e Desenvolvimento do Sistema Nervoso, Escola de Ciências da Saúde e da Vida, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil

P. Washbourne,     Institute of Neuroscience, University of Oregon, Eugene, OR, United States

Svante Winberg,     Department of Neuroscience, Uppsala University Uppsala, Sweden

Jiale Xu,     Department of Bioengineering and Therapeutic Sciences, Programs in Biological Sciences, Institute of Human Genetics, University of California, San Francisco, CA, United States

David Zada,     The Faculty of Life Sciences and the Multidisciplinary Brain Research Center, Bar-Ilan University, Ramat Gan, Israel

Irina V. Zhdanova,     BioChron LLC, Worcester, MA, United States

Acknowledgments

I would like to thank my family for coping with me staying up late for many nights working on this book, my students and colleagues whose brilliance and enthusiasm have been making my academic life a joy, and my anonymous referees whose expert opinion helped improve the quality of this book. I am also thankful for the wonderful academic environment and infrastructure the University of Toronto Mississauga has been providing to me, for the seamless support I have received from the staff of the Elsevier editorial office but particularly from Ms. Kristi Anderson and Dr. Natalie Farra, and, last but not least, for the diligent and dedicated work of my authors without which the publication of this book would not have been possible.

Introduction: The Zebrafish in behavioral and neural genetics

The last 10   years of the 20th century was declared The Decade of the Brain by a presidential proclamation (#6158) by US President George H. W. Bush, with the aim to enhance public awareness of the benefits to be derived from brain research (https://www.loc.gov/loc/brain/proclaim.html). The 21st century continued the theme. In 2013, US President Barak H. Obama announced the launch of the BRAIN (Brain Research through Advancing Innovative Neurotechnologies) Initiative (https://obamawhitehouse.archives.gov/BRAIN) whose goal is to revolutionize our understanding of the human brain, an effort in which both governmental agencies (including the National Institutes of Health (NIH) https://braininitiative.nih.gov/) as well as the private sector are participating. The brain, the most complex organ in our body, continues to fascinate politicians and the public alike, but it also remains to be the final frontier for science. Hundreds of millions of dollars are invested in brain research all over the word, and our knowledge is rapidly increasing as we are studying many aspects, including the macrostructure, the microstructure, the connectome, the physiology, the cell biology, the synaptic function, and the molecular biology and biochemistry of this fascinating organ. Several of these studies are conducted not with humans but with laboratory organisms that scientists argue could model numerous aspects of the human condition. Most in vivo studies of the 21st-century laboratory neuroscience employ a single model organism, the house mouse (Mus musculus domesticus). Focus on a single model organism is understandable. Empirical knowledge obtained by the myriad of independent studies conducted across a wide range of laboratories at multiple levels of the biological organization of this species may be fitted together with relative ease. What could another model organism, like the zebrafish, add to this already vast accumulated knowledge? This book, we hope, will provide a conclusive answer.

Most chapters of this book start with emphasizing the advantages of the zebrafish in behavioral and neural genetics as compared with other model organisms. Indeed, the zebrafish does have numerous excellent features that make it a useful species for neuroscience research. I will not reiterate these advantages here. Instead, I will draw the reader's attention to a point that provides yet another reason, a strong rationale, for the use of this species: the power of comparative approach. Simply put, a single laboratory organism with which scientists model and analyze the mechanisms of human functions or disorders may not be sufficient. Adding other species to this research should increase translational relevance, i.e., help us understand human brain function and dysfunction better. But why would a fish, separated from us and other mammals by about 400 million years of biological evolution, be useful for translational or biomedical research?

The most succinct way to explain the answer is by using Boolean algebra (Fig. 1). For this, consider what we know about biological evolution. We know that species are related to each other, and we also know that, on average, the more closely related they are, i.e., the less amount of time has passed since they had a common ancestor, the more similar they are. The problem is that we often do not know how to quantify, identify, and/or interpret similarity. If we were to be able to know for sure, for example, that a feature or biological function that appears similar between two species has a common evolutionary origin (a similarity we call homology in evolutionary biology), then we could pick and choose species that have features sufficiently similar to our own, just study those features in these species, and completely avoid the complications of having to work with humans. However, the problem is that quite often apparent similarity (even if we could precisely define and quantify its degree) does not guarantee evolutionary homology. Structures and functions that appear similar may have distinct evolutionary origins and thus, usually for this very reason, also distinct mechanisms that underly them. Take the example of the wings of insects, birds, or bats. How could we ascertain that the apparently similar features we identified between humans and another species are indeed evolutionarily homologous and mechanistically also similar? This is where the comparative approach comes in.

Looking for similarity according to Boolean logic is looking for an overlap between areas on Fig. 1. For example, consider the following. Assume that circle A represents all the molecular mechanisms that underly memory in humans and circle B the mechanisms that underly this same brain function in the mouse. The overlapping area represents the similarity. The problem is, of course, that we do not yet precisely know what this overlapping area is, i.e., we do not yet know all the mechanisms underlying memory in mice and humans and, very importantly, we also do not yet know all the mechanisms that do not underly memory in each of these species. So, how can we narrow our focus to the relevant mechanisms that really underly memory? If you add another species in which we can now identify mechanisms underlying memory (circle C), and look for the area that represents the overlap among all three of these species, we have identified the set of mechanisms common to all three of them. Given that species are related to each other, one will always be able to identify such overlap, i.e., common mechanisms. The identified commonalities not only represent the mechanisms that are more relevant to the given target phenotype, memory in our example, but they are also likely to be more fundamental to this target phenotype because they represent evolutionarily conserved aspects of the species studied. Evolutionarily conserved features, mechanisms, are likely to be the most important, core aspects of our biology. Studying the zebrafish and comparing the results we obtain for this species with those obtained for the mouse and human thus likely enhance our understanding of human brain function and dysfunction, i.e., likely increase translational relevance of research. To enable ourselves to make such comparisons, we need to accumulate data on this third species, zebrafish in our case. This book is a first attempt in summarizing some of the efforts to collect such data, within the field of neurobehavioral genetics.

Figure 1 Boolean logic of the comparative approach to explain why the use of multiple species increases translational relevance of research. Circles (A) human, (B) mouse, and (C) zebrafish represent mechanisms underlying a biological function, say, memory, for each of these species, respectively. The area that is the overlap among all three circles represents the mechanisms common to all three species. These mechanisms are the evolutionarily most conserved and thus most fundamental core aspects of the studied biological function.

This book is not meant to provide a comprehensive account of the methods and principles of neural and behavioral genetics of the zebrafish. This rapidly evolving field is already quite extensive (Fig. 2), and a single book cannot review all of its developments. Instead, our aim was to showcase the zebrafish by sampling some of the most interesting and fastest developing areas of neural and behavioral genetics research conducted with this species. The authors of the chapters are all experts of their respective fields, some of them are established senior investigators, others are rising stars, and, as is usual in science, they come from all over the world.

The chapters are organized into six sections. The first section briefly introduces the zebrafish and discusses questions about the natural ecology and ethology of this species as well as methods of maintenance and breeding of zebrafish. The focus of the second and third sections is the behavior of the fry and of the adult zebrafish, respectively. These sections review perceptual systems, motor function, motivation, and also some complex behaviors including social behavior, sleep, aggression, antipredatory behavior as well as different forms and aspects of learning and memory. Section four reviews few of the modern recombinant DNA methods developed for or adopted to the zebrafish, including transgenic reporter lines, and the CRISPR/Cas9 system, for example. Section five brings together the above discussed topics and reviews how they can be utilized to model and study different human disorders of the central nervous system using the zebrafish. This section includes chapters on modeling alcohol abuse–related diseases, Parkinson's disease, epilepsy, aging and sleep disorders, autism, and impulsivity. The last, sixth, section of the book is about data collection and bioinformatics-related questions and includes chapters on how to design and conduct high-throughput behavioral screens, what sort of software tools one can use for behavioral testing, how one can utilize bioinformatics resources for genetic analyses as well as for exploring the anatomy of the zebrafish brain.

Figure 2 The number of peer-reviewed publications per year as a function of time identified by searching the ISI Web of Knowledge database (all databases within this library search engine) using the simple terms zebrafish AND behavior (panel A), zebrafish AND brain (panel B), or zebrafish AND gene (panel C). Note the rapid increase of the number of these publications. Also note that the values obtained for the last year (year 2019) represent partial (incomplete count) results, as the data were obtained in the middle of that year (July).

The book is written for the expert neuroscientist who is interested in learning about zebrafish research and also for the zebrafish expert who would like to consider using genetic, neuroscience, and behavioral methods in his/her research. The book may also be useful for the undergraduate and graduate students interested in neural and behavioral genetics in general and those who would like to learn about novel approaches and directions in which this rapidly evolving and exciting field is heading.

Robert T. Gerlai

Section I

Introduction to Zebrafish: Natural habitat, ethology, and appropriate maintenance conditions

Outline

Chapter 1. The biotic and abiotic environment of zebrafish

Chapter 2. Maintenance and breeding of zebrafish, with some ethological and ecological considerations in mind

Chapter 1

The biotic and abiotic environment of zebrafish

David M. Parichy ¹ , and John H. Postlethwait ²       ¹ Department of Biology and Department of Cell Biology, University of Virginia, Charlottesville, VA, United States      ² Institute of Neuroscience, University of Oregon, Eugene, OR, United States

Abstract

The zebrafish is a premier laboratory model for developmental genetics and is used increasingly for ethology and behavioral neuroscience. A fuller appreciation of zebrafish behavior, and its underlying neurobiology, physiology, and genetics, demands an understanding of the species' ecology, past, and present. Studies of natural history are beginning to provide such context and reveal that zebrafish lives across diverse habitats, encompassing tremendous variation in temperature, water quality, altitude, and community composition. Integrating knowledge of biotic and abiotic environments with studies of behavior in the lab and field should provide new insights into the evolutionary origins and selective consequences of zebrafish behavioral phenotypes.

Keywords

Habitat; Natural history; Range; Temperature; Water quality; Zebrafish

Introduction

The zebrafish, Danio rerio, was recognized for its potential utility in developmental genetics (Chakrabarti et al., 1983; Kimmel, 1989; Grunwald and Eisen, 2002; Varga, 2018), but the species has become increasingly valuable for understanding behavior and the neurobiology underlying behavior (Kalueff et al., 2013; Gerlai, 2014; Orger and de Polavieja, 2017; Zabegalov et al., 2019). Viewed simply as a model system, the zebrafish can provide insights that may be generalizable to other vertebrates. Viewed as a species—with its own evolutionary history and selection regimes past and present—the zebrafish can inform our understanding of how behaviors, life history characteristics, morphology, developmental genetics, and physiology have been shaped in the wild. Whereas the former perspective requires only zebrafish and facilities to study them, the latter perspective requires the integration of such tools and resources with an intimate knowledge of the species' natural history.

To this end, we briefly review what is known of the zebrafish range, its evolutionary origins, and the abiotic and biotic environments in which it occurs. Given the fragmentary nature of this information and the limited scope of field studies thus far, our goal is not to paint a detailed portrait of how zebrafish behave in the natural world, but rather to collect some of the salient observations and to suggest avenues for future efforts.

Geographic distribution and phylogeny

The genus Danio presently comprises 26 valid species distributed across South Central and Southeast Asia (Tang et al., 2010; McCluskey and Postlethwait, 2015; Froese and Pauly, 2019). Encompassing a large geographical region, and studied in the wild by relatively few researchers, it seems likely that additional danios await discovery. Indeed, the number of recognized species has increased markedly in recent years (Fig. 1.1A).

The zebrafish itself is also distributed widely, with historical or more recent records from India, Nepal, Bangladesh, and Myanmar (Spence et al., 2006, 2007b, 2008; Engeszer et al., 2007; Whiteley et al., 2011; Arunachalam et al., 2013; Suriyampola et al., 2015; and references therein) (Fig. 1.1B). The fish may also occur in Pakistan (Daniels, 2002). The limits of the range are somewhat unclear given the paucity of sampling. Adding to this uncertainty, morphologically similar, striped danios also having ill-defined ranges, including Danio quagga and D. aff. kyathit (which may itself be D. quagga), could be mistaken for zebrafish in the eastern states of India, or vice versa in Myanmar (Fang, 1998; Quigley et al., 2005; Kullander et al., 2009; McCluskey and Postlethwait, 2015).

The phylogeny of zebrafish relative to other Danio species has been examined in several contexts, using different approaches and coming to different conclusions (Meyer et al., 1993, 1995; McClure, 1999; Parichy and Johnson, 2001; Sanger and McCune, 2002; Fang, 2003; Mayden et al., 2007; Fang et al., 2009; Tang et al., 2010).The most recent analyses using genome-wide data point to a D. rerio species group within which relationships are difficult to discern, owing at least in part to extensive introgression of chromosomal segments across phylogenetic lineages (McCluskey and Postlethwait, 2015). New opportunities for understanding the evolution of behavior and other traits may become apparent as genomes of new Danio species are placed within this developing framework.

Figure 1.1 Zebrafish evolution and range.(A) Cumulative numbers of Danio species, beginning with Danio rerio (arrow) (Hamilton, 1822). (B) Historical and recent sites at which D. rerio has been reported. Inset, Example adult D. rerio caught at a site in northeastern India (Engeszer et al., 2007). (C) Phylogenetic relationships of several Danio species, with the D. rerio species group shown in blue and red. Tree shown is only one of several recovered topologies. Images of Danio species with an emphasis on pigmentation and its evolution have been published (Quigley et al., 2004, 2005; Mills et al., 2007; Patterson et al., 2014; Spiewak et al., 2018; Lewis et al., 2019; Patterson and Parichy, 2019) in addition to species descriptions ( Fang, 1997b, 2000; 1997a; 1998; Fang and Kottelat, 1999, 2000; Roberts, 2007; Sen, 2007; Kullander and Fang, 2009b, 2009a; Kullander et al., 2009, 2015; Kullander, 2012, 2015; Kullander and Britz, 2015; Kullander and Noren, 2016 ). 

(C) After (McCluskey and Postlethwait, 2015).

Features of the abiotic and biotic environment

Table 1.1 summarizes observations of five studies plus unpublished field observations that documented zebrafish habitats in India and Bangladesh (McClure et al., 2006; Spence et al., 2006; Engeszer et al., 2007; Arunachalam et al., 2013; Suriyampola et al., 2015). Although the different studies emphasized different features of the environment, some generalities emerge. Some examples of sites at which zebrafish have been found are shown in Fig. 1.2.

Zebrafish have been found at elevations from just above sea level to over 1500   m. At these locations, zebrafish are most often found in shallow streams, backwaters, blind channels connected stably or intermittently to streams, or pools at stream edges. Substrates are commonly fine—mud, silt, or sand—but also can be pebbles, larger rock, or of mixed types. Not limited to natural bodies of water, zebrafish are found in rice paddies, irrigation ditches, and artificial ponds as well. Whether natural or artificial sites, zebrafish most often occupy still or slow moving water, sometimes adjacent to main channels having considerably faster flow. Banks are often well-vegetated, and waters frequently have overhanging and surface vegetation. Aquatic or submerged plants are often abundant. Vegetation presumably confers shade and considerable microenvironmental complexity, likely exploited for spawning, growth of larvae, and avoidance of predators. Indeed, still or nearly still waters with abundant plant material have been found to harbor apparently spawning zebrafish, and also juvenile zebrafish. Nevertheless, the life cycle of zebrafish in the wild and the relevance of laboratory observations to specific environments and behaviors in the field remain poorly understood (Spence et al., 2007a, 2007b; Engeszer et al., 2008; Suriyampola et al., 2015).

In keeping with structural and biological richness of their habitat, zebrafish in the wild have been found to eat animal material, especially insects of both terrestrial and aquatic origin, but also detritus, algae, and higher plant matter (McClure et al., 2006; Spence et al., 2007b; Arunachalam et al., 2013).

Waters containing zebrafish tend to be clear, although zebrafish can also be found occasionally in more turbid conditions, sometimes associated with rainfall or other disturbances. Details of light spectra and intensities in the aquatic environment—as experienced by zebrafish themselves—have yet to be documented unfortunately. Nevertheless, these data will be critical to interpreting behaviors and potential signals that are employed in agonistic interactions, courtship, shoaling, and predation avoidance (Rosenthal and Ryan, 2005; Rosenthal, 2007; Engeszer et al., 2008; Kalueff et al., 2013; Lewis et al., 2019; Zabegalov et al., 2019).

Although zebrafish in the laboratory are typically maintained at ∼28°C, they experience a considerable thermal range in nature, having been documented in waters from 12 to 39°C. It seems certain that some populations of fish experience colder conditions as well because high-altitude sites with the lowest recorded temperatures were surveyed during the summer months of June and July. Zebrafish have been found in waters with a pH from 5.9 to 9.2. These conditions suggest potentially interesting thermal and other physiological adaptations across populations. Understanding means and variances in these parameters across seasons, both within and among populations, will provide essential information for such efforts.

Table 1.1

a  ∼, Inferred from locality data through Google Earth.

b  In some instances fish may have been collected in pockets at stream edges.

c  nd, not determined.

d  A, mud or silt; B, sand; C, pebble, gravel or cobble; D, boulder or bedrock.

e  Still, no detectable flow; slow, <6   cm/s; medium, 6–12   cm/s; fast, >12   cm/s.

f  Clear, to bottom or ≥30   cm deep; intermediate, <30   cm deep; turbid, dirty, muddy or <5   cm deep.

g  A, Spence et al. (2006); B, McClure et al. (2006); C, Engeszer et al. (2007b); D, Arunachalam et al. (2013); E, Suriyampola et al. (2015); Postlethwait et al. field observations.

Figure 1.2 Examples of zebrafish habitat.(A–D) Stream with still pools and rice paddies in the Indian state of Meghalaya (sites 14–16 of Engezer et al., 2007). (A) Aerial view, recorded on November 2009 (Google Earth) showing locations and directions from which images in B, C, and D were taken during July 2006. N, North. (B) Small stream with well-vegetated banks. (C) Paddy fields above stream. (D) Still water with submerged vegetation adjacent to stream, flooded during the monsoon season, but dry in aerial view. Inset shows two zebrafish of a small shoal, adjacent to surface detritus (arrow, in main panel). (E) Small stream in West Bengal, India (site 3 of Engeszer et al., 2007). (F) Turbid pond with zebrafish and farmed fish in state of Orissa, India (site 24 of Engeszer et al., 2007). (G, H) Sites in Meghalaya, including a flooded road surface waters at 37°C (25.740610, 91.806707) and a small stream adjacent to a town (25.604767, 91.896118). Insets in G show a small shoal of zebrafish in shallow water as well as a single fish, with characteristic—and oddly conspicuous—spot of reflective iridophores that decorate the heads of zebrafish (arrow) (field observations, Postlethwait, Raman, Chatterjee, Dey, and Mylliemngap, unpublished).

Table 1.2

Aggregated from: Spence et al. (2006); Engeszer et al. (2007b); Arunachalam et al. (2013); Suriyampola et al. (2015)

Of particular interest for understanding the origins of zebrafish morphology and behavior are the other fishes with which it shares a habitat. A large number of species have now been documented to occur in the same bodies of water with zebrafish (Table 1.2; Fig. 1.3). These include potential competitors, like killifish (Aplocheilus), minnows (Esomus), barbs (Puntius), and others. Also present are potential predators of egg and larvae, including catfish (Mystus), loaches (Lepidocephalus), spiny eels (Mastacembelus), and torrent minnows (Psilorhynchus). Potential predators of adults include snakeheads (Channa), leaffish (Nandus), needlefish (Xenentodon), large catfish, and others. Beyond teleosts, it is likely that odonate larvae and other aquatic invertebrates prey upon zebrafish. Whether terrestrial animals, including wading or diving birds, take zebrafish remains unclear. Formal documentation of predatory and competitive interactions would provide valuable insights into the selection regime experienced by zebrafish, and the ecological consequences of behavioral variation across life cycle stages (Engeszer et al., 2007a; Buske and Gerlai, 2011).

Environmental changes to zebrafish habitat

As for all species, human impacts on zebrafish in the wild are likely substantial. Specific types of habitat degradation have been noted (Engeszer et al., 2007b; Arunachalam et al., 2013) and anecdotal observations from the field suggest that many sites formerly populated by zebrafish no longer support them. Given the wide geographic distribution of these fish, and the enormous wealth of knowledge provided by genomic, developmental, and other analyses, zebrafish would seem to be a valuable model for assessing the impacts of human activities on natural habitats and ecological and evolutionary responses to them. A sentinel role for zebrafish could provide insights into population and genetic consequences of direct human population encroachment; exposure to household, agricultural, or industrial products; and overall impacts of changing global and regional climates. Opportunities for such insightswill require those who study zebrafish in the wild to consistently publish high-resolution GPS data to allow field sites to be visited and revisited for years to come.

Figure 1.3 Species that co-occur with zebrafish.Only selected examples are presented. Color images of additional taxa can be found in Engeszer et al. (2007). Esomus, Oryzias, and other species might compete with zebrafish, whereas Lepidocephalus, Channa, Mystus, Barilius, and other species seem likely to be predators of zebrafish eggs, larvae, or adults. Large-bodied species of Danio and Devario often co-occur with smaller zebrafish; other small-bodied danio species occur typically further east than the known range of zebrafish (McCluskey and Postlethwait, 2015).

Conclusions

The zebrafish already offers an outstanding system for developmental biology and developmental neurobiology, evolution, genomics, and toxicology. As the species is increasingly adopted for behavioral neuroscience or ethological investigations, a deeper understandingof its natural world will surely provide valuable context and may lead to insights that would not otherwise be apparent. Despite the geographical distance of wild zebrafish from the majority of labs that use the species, the studies reviewed here, and the authors' own experiences, indicate that field work is both tractable and illuminating. When done collaboratively, such efforts also can lead to valuable scientific and cultural bridges for investigators and trainees across the globe.

Acknowledgments

DMP and research in his laboratory is supported by NIH R35 GM122571. JHP acknowledges support of NIH grant R01 OD011116 and the efforts of Prof. Rajiva Raman (Cytogenetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi, India) and Prof. Anupam Chatterjee (Head of the Department of Biotechnology, North Eastern Hill University, Shillong, Meghalaya, India), who facilitated zebrafish collections and lab support, along with the skillful help of Sankhadip Dey and Brandon Keith Mylliemngap (North-Eastern Hill University, Shillong, Meghalaya, India) and Prof. Manfred Schartl (University of Würzburg, Germany).

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Chapter 2

Maintenance and breeding of zebrafish, with some ethological and ecological considerations in mind

Benjamin Tsang ¹ , Rida Ansari ² , ³ , and Robert T. Gerlai ¹       ¹ Department of Psychology, University of Toronto Mississauga, Mississauga, ON, Canada      ² Department of Cell and Systems Biology, University of Toronto, Toronto, ON, Canada      ³ Neuroscience and Mental Health Program, Hospital for Sick Children, Peter Gilgan Centre for Research and Learning, Toronto, ON, Canada

Abstract

The popularity of zebrafish is exponentially increasing. Unlike in the past, the zebrafish now is a subject of investigation by scientists of practically every subfield of biology, including psychopharmacology and behavior genetics. Simply put, the zebrafish is no longer considered a simple egg-producing tube for efficient delivery of thousands of embryos to be studied only by developmental biologists. With the broadening interest, come numerous challenges. One of the most fundamental of which is how to maintain and breed zebrafish. Here we consider some questions of zebrafish maintenance and breeding, and provide a few examples of what we argue would be important issues where some change may be required. We emphasize that systematic parametric analyses of environmental factors are needed to optimize the laboratory environment for the zebrafish. Last, we argue that understanding the ecology and ethology of the zebrafish is important for establishing proper maintenance and breeding of this species.

Keywords

Breeding; Learning and memory; pH; Salinity; Salt composition; Social behavior; Stress; Water chemistry; Zebrafish maintenance

Introduction

Zebrafish use in basic science has grown at a rapid pace. Initiated about 4.5 decades ago by Dr. George Streisinger from the University of Oregon, zebrafish research has quickly taken the scientific community by storm. The zebrafish has seen an exponential growth as an animal model for a wide range of scientific inquiries. From developmental biology to molecular genetics, the number of researchers adopting the zebrafish has been exponentially increasing as a result of this species' amenability to a number of genetic and pharmacological manipulations, in addition to its attractive practical features including its small size and highly prolific nature. Unfortunately, the study of the fundamental basics of how to keep and raise this fish has not seen such exponential growth, which has led to potential suboptimal industry standards, and/or the diversification of husbandry methods based upon individual laboratory practices, abilities, or personal biases.

Perhaps one of the reasons for the absence of systematic studies on zebrafish maintenance is that the past 5   decades of zebrafish research has been dominated by developmental biology and molecular genetic studies (Carney and Mosimann, 2018; Fuentes et al., 2018; Patton and Tobin, 2019). Many of these studies asked questions about fundamental developmental processes ;focusing on only the first few hours or days of zebrafish ontogenesis and investigated genes with major effects. For these studies, efficient production of a large number of embryos was the major goal, and thus the industry standards of zebrafish maintenance were developed accordingly. However, as we have mentioned, the past decade has seen an explosive growth of zebrafish studies representing practically every scientific subfields of biology, from cancer research to animal cognition (Cayuela et al., 2019; Adams and Kafaligonul, 2018; Choo and Shaikh, 2018; Roper and Tanguay, 2018; Torraca and Mostowy, 2018; Dooley and Zon, 2000; Amatruda et al., 2002). These studies often utilize not only embryonic or larval but also adult zebrafish. Furthermore, these studies often investigate biological processes that are expected to be influenced by numerous genes with minor effects and/or by several environmental factors, including those that are affected by the methods of maintenance and breeding of zebrafish in the laboratory. Appreciation of such factors is particularly crucial for neuroscientists and behavioral scientists who investigate the functioning of one of the most plastic organs of the vertebrate body, the brain. Thus, optimization of environmental conditions in the laboratory has become a relevant question.

Our survey of the literature and online information revealed substantial variation in maintenance methods and environmental conditions employed across laboratories, and occasionally even within the same laboratory (Tsang et al., 2017), an issue that was also discussed at a recent NIH-organized symposium titled Zebrafish and Other Fish Models: Extrinsic Environmental Factors for Rigorous Experiments and Reproducible Results (https://orip.nih.gov/zebrafish-and-other-fish-models-extrinsic-environmental-factors-rigorous-experiments-and).

In this chapter, we will discuss some of the current methodologies employed in zebrafish husbandry and emphasize that, although zebrafish have been maintained in the laboratory for decades, systematic and parametric analyses of the optimal environmental conditions under which this species should be kept are often lacking. We also argue that for developing such optimized maintenance and breeding conditions, one needs to consider the natural conditions in which these fish thrive. But not everyone agrees. There are several considerations for setting up a zebrafish facility and for establishing the environmental parameters for the maintenance and breeding of these fish. Some of these considerations include efficiency, cost, footprint, ease of facility maintenance. In fact, some believe that these considerations argue against modeling the laboratory environment after how zebrafish live in nature. Yet, others, including us, explain that consideration of the ethology (the natural behavioral aspects of the species) and the ecology (the natural environmental conditions in which the species evolved and lives) is