Zebrafish are being developed as a model for complex human brain disorders by characterizing their behavioral phenotypes. The dissertation aims to 1) quantify zebrafish behaviors exposed to treatments modifying affective, social and cognitive domains, 2) develop automated behavioral quantification techniques, and 3) identify new phenotypic features using 3D trajectory reconstructions. Results showed zebrafish display anxiety-like behaviors in response to anxiogenic/anxiolytic drugs similarly to rodents and humans. Hallucinogenic drugs modified exploration and movement patterns. Automated techniques were developed and new measures from trajectories distinguished treatments. Overall, zebrafish were validated as a translational model for neurobehavioral research.

1. DEVELOPING ZEBRAFISH MODELS OF
COMPLEX PHENOTYPES RELEVANTTO
HUMAN BRAIN DISORDERS
Jonathan M. Cachat, MS
Committee
AllanV. Kalueff, Ph.D., Director
Jill Daniel, Ph.D.
David Corey, Ph.D
Benjamin Hall, Ph.D.
Neuroscience Program
School of Science and Engineering

2. Outline
• Challenges in Neurobehavioral Research
• Zebrafish (Danio rerio)
• Problem Statement
• Central Hypothesis, Research Strategy and SpecificAims
• Results
• Conclusions
• TranslationalValue
• Future Directions of Research

3. Challenges in Neurobehavioral Research
Use models to elucidate etiological and pathological
mechanisms of human brain disorders
(Gerlai, 2012; Burne et al., 2011; Morris, 2009; Sison et al., 2006)
• Developing sensitive, high-throughput, cost effective in-vivo
screening assays
• Standardizing and enhancing methodologies for objective
acquisition and analysis of behavioral data

4. Zebrafish (Danio rerio)
(Cachat et al., 2010)

5. 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)

6. 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

7. 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

8. ResearchApproach

9. 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

10. Design of BehavioralTests
Specific Aim 1
Characterize and Quantify Behavioral Phenotypes of Zebrafish
exposed to ExperimentalTreatments in
Affective, Social and Cognitive Domains

11. Specific Aim 2
Specific Aim 1
Characterize and Quantify Behavioral Phenotypes exposed to Experimental
Treatments Modifying Affective, Social and Cognitive Domains
DevelopAutomated Quantification
Techniques of Behavioral Endpoints

12. Specific Aim 3
Specific Aim 1
Characterize and Quantify Behavioral Phenotypes exposed to Experimental
Treatments Modifying Affective, Social and Cognitive Domains
Specific Aim 2
Develop Automated Behavioral Quantification of Phenotypic Measures
Identify New
Phenotypic Features
Evaluating 3D
Trajectory
Reconstructions

13. Modeling Affective Phenotypes
Ethologically Relevant Stimuli
• Alarm Pheromone Exposure
• Predator Exposure
How do zebrafish display anxiety/fear-like behaviors?
NovelTankTest
• 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

14. Primary Endpoints
NovelTankTest
• Latency to Upper Half, s
• Transitions to Upper Half
• Time Spent in Upper Half
• Erratic Movements
• Freezing Bouts
• Freezing Duration, s

15. Predator Exposure
(Cachat et al., 2010)

16. Alarm Pheromone
Representative 2D Swim Traces
(Cachat et al., 2011)

17. Summary of Results I
Establishing Anxiogenic
Profile in NovelTankTest
PharmacologicalTreatments

18. PharmacologicalTreatments
• Caffeine
• Fluoxetine
• Ethanol
• Nicotine
• Cocaine
• Morphine
• EthanolWithdrawal
• CaffeineWithdrawal
• MorphineWithdrawal
• 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?

19. Caffeine
Representative 2D Swim Traces
(Cachat et al., 2011)

20. 3DTrajectory Reconstructions
Wild-Type Control
Alarm Pheromone
Caffeine
(Cachat et al., 2011)

21. Fluoxetine
Representative
2D Swim Traces
(Cachat et al., 2011)

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

23. Nicotine
Representative
2D Swim Traces
(Cachat et al., 2011)

24. Wild-Type Control
Chronic Ethanol
Acute Nicotine
Chronic Fluoxetine
(Cachat et al., 2011)

25. Summary of Results II
Establishing an Anxiolytic Profile
in NovelTankTest
Reproducing Anxiogenic
Profile in NovelTankTest
AcuteCaffeine
Acute
Pentobarbital
AcuteFluoxetine
ChronicFluoxetine
AcuteEthanol
ChronicEthanol
AcuteNicotine
- - -
-
- -
- - - -
- - -
- - -
na na na
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

26. Chronic Morphine
Representative
2D Swim Traces
(Cachat et al., 2011)

27. Withdrawal
(Cachat et al., 2011)

28. Wild-Type Control
Ethanol Withdrawal
Chronic Morphine
Repeated Morphine
Withdrawal
(Cachat et al., 2011)

29. Summary of Results III
Reproducing Anxiolytic &
Anxiogenic Profile in NovelTank
Test
Cocaine(1mg/L)
Cocaine(12.5mg/L)
Cocaine(25mg/L)
AcuteMorphine
ChronicMorphine
Caffeine
Withdrawal
Repeated
Morphine
Withdrawal
EthanolWithdrawal
- - - -
-
- - - - - -
- - - - -
- - - - - -
- - -
na na na na -
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

30. Modeling Affective Phenotypes
How do zebrafish display anxiety/fear-like behaviors?
Results: In the NovelTankTest,
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
3DTrajectory Reconstructions reveal the spatial and temporal dynamics of these responses

31. TranslationalValue
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

32. 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)

33. Correlation Observable in 3D Reconstructions
(Cachat et al., 2011)

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

35. Using 3D Reconstructions to Optimize Automated
Techniques

36. Modeling Hallucinogenic Phenotypes
Are zebrafish sensitive to hallucinogenic compounds? How do these drugs
alter behaviors in affective, social and cognitive domains?
NovelTankTest, Open FieldTest, 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

37. HallucinogenicTreatments
• 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

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

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

40. Shoaling and Social Preference
(Grossman et al., 2010; Cachat, 2013)
LSD (250 µg/L, 20 min) Ibogaine (10, 20 mg/L, 20 min)

42. Modeling Hallucinogenic Phenotypes
Are zebrafish sensitive to hallucinogenic compounds? How do these drugs
alter behaviors in affective, social and cognitive domains?
Answer:
Big Question:
NovelTankTest – mixed behavioral & physiological profile, largely insignificant
Light-Dark Box – increase preference for white chamber compared to matched controls, with LSD increasing
cortisol
Open FieldTest – primary endpoints insignificantly altered in LSD and Ibogaine treated fish compared to
controls
Social Domains – no effects on social preference, decreased shoal cohesion
3DTrajectories – 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.

43. Identify New Phenotypic Features
using 3DTrajectory ReconstructionsSpecific Aim 3

44. Reveal Phenotypic Differences

45. Identification of Novel Phenotypes

46. Fractal d
Fractal d = 1.163
Fractal d = 1.169
Fractal d = 1.94
Segmentation
Smoothed Data

47. Arena Segmentation
Preliminary results suggest that
feature sets based on new arena
segmentations could be used to
discriminate between treatments

48. Future Directions of Research
• Zebrafish Movement Database
• Application ofTrajectory & Movement Pattern AnalysisTechniques
• Customized Analysis IntrinsicThresholds
• Multi-Scale Straightness Index (MSSI)

49. Summary of Dissertation
• ValidatedZebrafish asTranslationally Relevant Model for Neurobehavioral
Research
• Replicating Previous Findings
• Enhancing Phenotypic Characterization
• Paralleling Rodent and Clinical Profiles
• Introduced Zebrafish as model for Hallucinogenic Drug Action
• EstablishedApproaches toAchieve Automate Analysis of Zebrafish Behavior
• Developed Innovative 3DTrajectory Reconstructions and New Endpoints with
potential to Distinguishing ExperimentalTreatments

50. Acknowledgements
• Kalueff Lab
• Members of Dissertation
Committee
• Dr. Jill Daniel
• Dr. David Corey
• Dr. Benjamin Hall
• Tulane University Neuroscience
Program
• Dr. JefferyTasker
• Sherrie Calogero
• Collaborators
• Noldus InformationTechnologies
• 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 ChapterTravelAward