Developing Zebrafish Models of Complex Phenotypes Relevant to Human Brain Disorders
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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.
Developing Zebrafish Models of Complex Phenotypes Relevant to Human Brain Disorders
- 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
- 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
- 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