Developing Zebrafish Models of Complex Phenotypes Relevant to Human Brain Disorders

DEVELOPING ZEBRAFISH MODELS OF COMPLEX PHENOTYPES RELEVANT TO

HUMAN BRAIN DISORDERS

Jonathan M. Cachat, MS

CommitteeAllan V. Kalueff, Ph.D., Director

Jill Daniel, Ph.D.David Corey, Ph.D

Benjamin Hall, Ph.D.

Neuroscience ProgramSchool of Science and Engineering

Outline• Challenges in Neurobehavioral Research

• Zebrafish (Danio rerio)

• Problem Statement

• Central Hypothesis, Research Strategy and Specific Aims

• Results

• Conclusions

• Translational Value

• Future Directions of Research

Challenges in Neurobehavioral Research

Use models to elucidate etiological and pathologicalmechanisms of human brain disorders

(Gerlai, 2012; Burne et al., 2011; Morris, 2009; Sison et al., 2006)

• Developing sensitive, high-throughput, cost effective in-vivoscreening assays

• Standardizing and enhancing methodologies for objective acquisition and analysis of behavioral data

Zebrafish (Danio rerio)

(Cachat et al., 2010)

Zebrafish Genome• Genome duplication event

• Paralogous genes & sub-functionalization

• Advantageous (i.e. Sonic hedgehog knockdown)

• Zebrafish possess high nucleotide sequence homology (70-80%) with that of human genes.

• Functionally relevant as the amino acid sequence of zebrafish proteins (60-90% sequence homology) especially at the functionally relevant catalytic or ligand binding domains (approaching 100% sequence homology).

(Dooley, 2000; Renier et al., 2007; Reimers et al., 2004; Gerlai, 2011; Lillesaar, 2011)

Central Nervous System & HPI-Axis

(Lillesar, 2011; Mueller, 2012; Panula, 2010; Alsop, 2009; Cachat, 2010)

Homologous Brain Regions Relevant to Affective Research• Amygdala – Dm, medial zone of dorsal telencephalon• Hippocampus

Problem Statement

Adult zebrafish behavioral phenotypes are largely uncharacterized due to a lack of available, validated behavioral test paradigms

(Agid et al., 2007; Blaser et al., 2012; Luca et al., 2012; Savio et al., 2012; Sison et al., 2006)

The overarching goal of this dissertation is to advance the characterization of adult zebrafish behaviors, and progress comprehensive quantification of phenotypic profiles translationally relevant to

neuropsychiatric disorders.

Dissecting adult zebrafish behavior is a necessary process before targeted genetic or molecular high-throughput screens can be confidently hypothesized and performed

Research Approach

Central Hypothesis• Zebrafish represent a sensitive, highly translational, evolutionarily related

organisms that can be used to model endophenotypes of human brain disorders

I expect that

• A integrative approach to quantify behavioral and physiological phenotypes in zebrafish following several psychotropic treatments result profiles analogous to those observed in rodents and humans

• Using automated video-tracking technologies will enable objective detection and dissection of behavioral profiles in adult zebrafish, as well as 3D reconstructions of swim paths

• Increasing the granularity, data density collected per fish will enable application of data-mining and detection of new dependent variables that could potentially be used to predict the mechanism of action in novel or poorly characterized psychotropic compounds

Design of Behavioral Tests

Specific Aim 1Characterize and Quantify Behavioral Phenotypes of Zebrafish exposed to Experimental Treatments in Affective, Social and Cognitive Domains

Specific Aim 2

Specific Aim 1Characterize and Quantify Behavioral Phenotypes exposed to Experimental Treatments Modifying Affective, Social and Cognitive Domains

Develop Automated Quantification Techniques of Behavioral Endpoints

Specific Aim 3

Specific Aim 1Characterize and Quantify Behavioral Phenotypes exposed to Experimental Treatments Modifying Affective, Social and Cognitive Domains

Specific Aim 2Develop Automated Behavioral Quantification of Phenotypic Measures

Identify New Phenotypic Features

Evaluating 3D Trajectory

Reconstructions

Modeling Affective Phenotypes

Ethologically Relevant Stimuli

• Alarm Pheromone Exposure

• Predator Exposure

How do zebrafish display anxiety/fear-like behaviors?

Novel Tank Test • Manual (observation, event-based) and automated behavioral quantification• Analysis of behavior in 3D trajectory reconstructions

Approach: Behavior

Big Question:

Pharmacological Treatments

• Putative Anxiogenic and Anxiolytic Drugs

Physiology

Whole-body Cortisol Concentrations

Primary Endpoints

Novel Tank Test

• Latency to Upper Half, s

• Transitions to Upper Half

• Time Spent in Upper Half

• Erratic Movements

• Freezing Bouts

• Freezing Duration, s

Predator Exposure

(Cachat et al., 2010)

Alarm Pheromone

Representative 2D Swim Traces

(Cachat et al., 2011)

Summary of Results I

Establishing Anxiogenic Profile in Novel Tank Test

Pharmacological Treatments

Pharmacological Treatments

• Caffeine

• Fluoxetine

• Ethanol

• Nicotine

• Cocaine

• Morphine

• Ethanol Withdrawal

• Caffeine Withdrawal

• Morphine Withdrawal

• Known to evoke anxiogenic or anxiolytic behavioral responses in humans and rodents

• Can zebrafish be used to model phenotypes relevant to pharmacogenic anxiety and drug abuse related syndromes?

Caffeine

Representative 2D Swim Traces

(Cachat et al., 2011)

3D Trajectory Reconstructions

Wild-Type Control

Alarm Pheromone

Caffeine

(Cachat et al., 2011)

Fluoxetine

Representative

2D Swim Traces

(Cachat et al., 2011)

Ethanol

(Egan et. al., 2009; Cachat et al., 2011)

Nicotine

Representative

2D Swim Traces

(Cachat et al., 2011)

Wild-Type Control

Chronic Ethanol

Acute Nicotine

Chronic Fluoxetine

(Cachat et al., 2011)

Summary of Results II

Establishing an Anxiolytic Profilein Novel Tank Test

Reproducing Anxiogenic Profile in Novel Tank Test

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Pharmacological Treatments

Latency to upper half, s

Transitions to upper half

Erratic movements

Freezing bouts

Freezing duration, s

Cortisol Concentration

Time in upper half, s

Chronic Morphine

Representative

2D Swim Traces

(Cachat et al., 2011)

Withdrawal

(Cachat et al., 2011)

Wild-Type Control

Ethanol Withdrawal

Chronic Morphine

Repeated Morphine Withdrawal

(Cachat et al., 2011)

Summary of Results III

Reproducing Anxiolytic & Anxiogenic Profile in Novel Tank Test

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Drug WithdrawalDrugs of Abuse

Freezing bouts

Freezing duration, s

Cortisol Concentration

Latency to upper half, s

Transitions to upper half

Time in upper half, s

Erratic movements

Modeling Affective Phenotypes

How do zebrafish display anxiety/fear-like behaviors?

Results: In the Novel Tank Test,

Question:

A high anxiety behavioral profile in zebrafish is represented by:• Decreased exploration throughout environment• Increase in freezing throughout novel tank test• Short-lived, erratic movements • Stress-axis activation as measured by cortisol concentrations

A low anxiety profile is reflected by an inverse of this behavioral profile

3D Trajectory Reconstructions reveal the spatial and temporal dynamics of these responses

Translational Value

Zebrafish behavioral response following ethological and pharmacological treatments paralleled the changes observed in the affective domain of rodents and humans.

• Including dose and duration specific effects

Primary endpoints of novel tank are able to reliably distinguish between strong anxiogenic and anxiolytic phenotypes

Can detection of these phenotypes be automated for NTT?

Automation

Specific Aim 2

Distance, m Velocity, m/s TurnAngle, ° TurnBias, °/s

Control 0.0015 0.0460 0.0268 0.80

Anxiogenic 0.0011 0.0321 0.0776 2.33

Anxiolytic 0.0011 0.0319 0.2509 7.52

Average 0.0012 0.0367 0.1184 3.55

Control 0.0034 0.1019 5.0300 150.75

Anxiogenic 0.0023 0.0677 3.0255 90.67

Anxiolytic 0.0016 0.0493 0.8654 25.94

Average 0.0024 0.0730 2.9736 89.12

Control 0.0000 0.0004 -0.1433 -4.29

Anxiogenic 0.0001 0.0025 0.3319 9.95

Average 0.0000 0.0014 0.0943 2.83

Erratic

Freezing

Automated Movement Parameter

Swimming

Behavioral State (Manually Recorded)

Behavioral tests designed precisely integrate manual (observation, event-based scoring) and automated

quantification within individual spatiotemporal data

Manual, Event based & Automated

Manual Observation & Manual, Event-Based

Develop Automated Quantification Techniques of Behavioral Endpoints

(Cachat et al., 2011)

Correlation Observable in 3D Reconstructions

(Cachat et al., 2011)

Automated Detection of Complex Behavior(Cachat et al., 2011)

Using 3D Reconstructions to Optimize Automated Techniques

Modeling Hallucinogenic Phenotypes

Are zebrafish sensitive to hallucinogenic compounds? How do these drugs alter behaviors in affective, social and cognitive domains?

Novel Tank Test, Open Field Test, Light-Dark Box, Shoaling, Social Preference Tests• Manual (observational, event-based) and automated behavioral quantification• Analysis of behavior in 3D trajectory reconstructions

Approach: Behavior

Big Question:

Physiology

Whole-body Cortisol Concentrations

Hallucinogenic Treatments• Lysergic acid diethylamide (LSD)

• 3, 4-Methylenedioxymethylamphetamine (MDMA)

• Ibogaine

• LSD = 1.0964

• MDMA = 1.1293

• Most profound effects on rodents and humans

• Never been examined before in zebrafish

• Recent resurgence of interest in hallucinogenic drug action for use in clinical therapy

Novel Tank Test

MDMA

(Grossman et al., 2010; Stewart, 2011; Cachat, 2013)

Light-Dark Box and Open-Field Test(Grossman et al., 2010; Cachat, 2013)

Shoaling and Social Preference

(Grossman et al., 2010; Cachat, 2013)

LSD (250 µg/L, 20 min) Ibogaine (10, 20 mg/L, 20 min)

Modeling Hallucinogenic Phenotypes

Are zebrafish sensitive to hallucinogenic compounds? How do these drugs alter behaviors in affective, social and cognitive domains?

Answer:

Big Question:

Novel Tank Test – mixed behavioral & physiological profile, largely insignificant

Light-Dark Box – increase preference for white chamber compared to matched controls, with LSD increasing cortisol

Open Field Test – primary endpoints insignificantly altered in LSD and Ibogaine treated fish compared to controls

Social Domains – no effects on social preference, decreased shoal cohesion

3D Trajectories – strong modifications on zebrafish exploration and movement

Zebrafish are sensitive to hallucinogenic compounds, but phenotypic domains unclear with primary endpoints in behavioral tests – however trajectories illustrate evident differences, suggesting that novel measures are necessary to provide detailed characterization.

Identify New Phenotypic Features using 3D Trajectory ReconstructionsSpecific Aim 3

Reveal Phenotypic Differences

Identification of Novel Phenotypes

Fractal d

Fractal d = 1.163

Fractal d = 1.169

Fractal d = 1.94

Segmentation

Smoothed Data

Arena Segmentation

Preliminary results suggest that feature sets based on new arena segmentations could be used to

discriminate between treatments

Future Directions of Research

• Zebrafish Movement Database

• Application of Trajectory & Movement Pattern Analysis Techniques• Customized Analysis Intrinsic Thresholds

• Multi-Scale Straightness Index (MSSI)

Summary of Dissertation• Validated Zebrafish as Translationally Relevant Model for Neurobehavioral

Research• Replicating Previous Findings

• Enhancing Phenotypic Characterization

• Paralleling Rodent and Clinical Profiles

• Introduced Zebrafish as model for Hallucinogenic Drug Action

• Established Approaches to Achieve Automate Analysis of Zebrafish Behavior

• Developed Innovative 3D Trajectory Reconstructions and New Endpoints with potential to Distinguishing Experimental Treatments

Acknowledgements• Kalueff Lab

• Members of Dissertation Committee• Dr. Jill Daniel

• Dr. David Corey

• Dr. Benjamin Hall

• Tulane University Neuroscience Program• Dr. Jeffery Tasker

• Sherrie Calogero

• Collaborators• Noldus Information Technologies

• University of Zurich –GIS Department

• Dr. Ramgopal Mettu, TU Comp Sci

• Grants• NIH

• Louisiana Board of Regents

• Tulane University

• NIDA

• Fellowships/Awards• NSF

• SfN GNOSN Chapter Travel Award