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Workshop on Behavioral models in zebrafish

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Workshop on the Universidad Veracruzana, as part of the commemoration of tge 25th Anniversary of the Neuroethology Program

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Workshop on Behavioral models in zebrafish

  1. 1. Workshop on Behavioral models in zebrafish Lecture 1: First principles
  2. 2. First day schedule ● Ethoexperimental approaches to zebrafish behavior and relationship to natural history ● Anxiety assays ● Aggression and social behavior ● Practical issues 1: General care and manipulation ● Practical issues 2: Anesthesia and drug administration ● Practical issues 3: Pseudoreplication and experimental design
  3. 3. Why zebrafish? Kokel D , and Peterson R T Briefings in Functional Genomics and Proteomics 2008;7:483-490 ● Vertebrate species ● Intermediate physiological and behavioral complexity ● Medium-to-high troughput ● Advantages as model organism: – Reproduce quickly and abundantly – Transparent embryos – Rapid development – Fully sequenced genome (85% homology with – humans) – Ease of genetic manipulation – Low-cost model (1 x 1000 vs. rodents)
  4. 4. Applications of behavioral models and tests ● Drug discovery and development ● Pharmacogenetic models ● Validation research (construct validity, “a-theoretical” approaches using Big Data, etc) ● Environmental toxicology
  5. 5. Ethoexperimental approaches ● Blanchard & Blanchard (1988): In the context of Animal Psychology, discovery will only be possible if the (then dominant) experimental approach has an ethological twist – Rigorous experimental control of intervening variables – Inspiration on findings from ethology and behavioral biology ● Great influence in the field of anxiety research ● In order to develop good tests and models in zebrafish, we need to be familiar with its ecology
  6. 6. The zebrafish (Danio rerio): General aspects ● Cyprinidae ● Small size (around 2 to 4 cm) ● Native from Southeast Asia ● Live in shoals ● Eggs develop externally ● Transparent embryos
  7. 7. Natural history ♂ ♀ http://dx.doi.org/10.7554/eLife.05635.002
  8. 8. Natural history http://dx.doi.org/10.7554/eLife.05635.003 Competitors Predators
  9. 9. Natural history Common kingfisher Indian pond heron
  10. 10. Consequences for behavior ● Most of their food come from the water column, mainly eating zooplankton and aquatic insects… ● ...BUT predators also come from above! ● Olfaction, vision, and motion detection via the lateral line system help zebrafish perceive their local environment and evade potential predators – Movement in the surrounding water is detected by the lateral line, which can detect small changes in pressure in the immediate environment. ● Zebrafish respond to a broad range of chemical cues detected by the olfactory bulb.
  11. 11. Behavioral assays
  12. 12. A caveat: Models vs. tests ● Strictly speaking, the majority of protocols used to test a given domain (e.g., anxiety, aggression, etc.) are tests or assays, not true models ● No reference to etiology; most tests attempt to measure a given construct in different contexts – Normal vs. mutant animals – Normal vs. lesioned animals – Behavioral extremes – Drug effects – Toxicology – … ● A model should make reference to a specific, real disorder (e.g., GAD, MDD, schizophrenia); a test is broader, but its utility in finding specific treatments is low
  13. 13. Anxiety assays ● Anxiety vs. fear: certain vs. uncertain threat ● The objective of these assays is to assess state anxiety; not to be confused with a model for anxiety, which needs better knowledge of etiology ● Novelty-based vs. conflict-based
  14. 14. A novelty-based assay: NTT ● One of the most widely used tests in zebrafish – Created by Ed Levin, popularized by Allan Kalueff ● Ethological rationale: predator risk vs. feeding opportunities ● “Diving” response, when associated with freezing, as an index of anxiety – Stimulus control (Rachel Blaser): diving response is controlled mainly by top avoidance
  15. 15. Predictive validity
  16. 16. Is there pharmacological isomorphism? Treatment Bottom-dwelling Erratic swimming Freezing Chronic SSRIs ↓ ↓ ? BZDs ↓ ? ? iMAOs ↓ 0 0 Barbiturates ↓ 0 0 Buspirone ↓ 0 ↓
  17. 17. Issues ● Selective reporting in the literature makes for confusing interpretation of endpoints – E.g.: less time on bottom interpreted as less anxiety (e.g., Egan et al., 2009) or as hallucination-like state (e.g., Neelkantan et al., 2013) ● Less sensitive to treatments than the LDT (Kysil et al., 2017)
  18. 18. A conflict-based assay: The light/dark test ● One of the most widely used tests in zebrafish – Created by Rosana Matiolli, popularized by me ● Ethological rationale: Crypsis ● Preference for dark substratum*, associated with risk assessment, thigmotaxis, erratic swimming, and freezing – Stimulus control (Blaser + me): preference is controlled by an approach-avoidance conflict
  19. 19. Is there pharmacological isomorphism? Treatment Scototaxis Risk assessment Thigmotaxis Erratic swimming Freezing Chronic SSRIs ↓ ↓ ↓ 0 0 BZDs ↓ 0 0 ↓ ↓ iMAOs 0 0 0 0 0 Barbiturates ? ? ? ? ? Buspirone ↓ ↓ ↓ 0 ↓
  20. 20. Comparing between NTT and LDT ● Kysil et al. (2017): Comparison between both tests – Metanalysis of drug effects suggest that LDT is more sensitive than NTT – No difference in cortisol response produced by both tests – Moderate correlation (r² = 0.55) between time on white and time on top ● Maximino et al. (2012): Differing stimulus control suggests that both tests measure different things ● Complementary use of both tests is suggested
  21. 21. Social behavior
  22. 22. Shoaling and social preference ● In shoaling tests, animals are tested in the same tank, and endpoints usually include interfish distance (and variants) ● In social preference, the preference of a focal animal is observed
  23. 23. Shoaling and social preference ● Multiple functions of shoaling in the wild (Pitcher & Parrish, 1993) – Better orientation – Synchronized hunting – Predator confusion – Reduced risk of being found ● Shoaling is motivated behavior (Al-Imari & Gerlai, 2008), but fear-inducing stimuli increase shoaling (Speedie & Gerlai, 2008) ● Social preference is increased by treatment with anxiolytic drugs (buspirone and WIN 55,212) (Barba-Escobedo and Gould, 2012)
  24. 24. Aggression ● With sociality comes aggressive behavior – Establishment of dominance relationships and hierarchies – Competition for key resources (food, shelter, mates, territories) ● Quantification of zebrafish aggression made in dyads or with the MIA
  25. 25. Aggression ● Advantages of dyads: full repertoire; fight resolution (winner vs. loser) ● Advantages of MIA: no injuries, timing
  26. 26. Practical issues
  27. 27. Issue 1: General care ● Not many guidelines on the use of fish ● Based on Lawrence (2007), Brazilian law, and own experience: – ALWAYS house animals in groups – Environmental enrichment is not the standard, but is desirable due to welfare issues – Tanks vary widely, from commercial, lab-specific (e.g., Tecniplast) to housing animals in plastic containers ● Always make sure tank material is BPA-free!
  28. 28. The importance of random housing ● If tanks are positioned in a shelf, animals should be randomly drawn from different shelves for each condition ● OR at least all animals from all groups need to be taken from the same tank ● Reduces performance biases due to, e.g., water quality
  29. 29. Water quality monitoring is the main QC parameter in zebrafish research ● Daily monitoring of – Temperature – pH – Conductivity ● Weekly monitoring of – Ammonia – Nitrite – Nitrate – Hardness ● Always check dissolved oxygen and nitrite before experiments
  30. 30. Water quality parameters Parameter Desired value Tolerable amplitude Temperature 28 °C 27 – 29 °C pH 7,5 7,3 – 7,7 Conductivity 1200 µS 1150 – 1250 Ammonia 0 ppm 0 – 2 ppm Nitrite 0 ppm 0 – 2 ppm Nitrate 0 – 10 ppm 0 – 100 ppm Hardness > 80 ppm CaCO3 80 – 300 ppm CaCO3 Dissolved oxygen > 5 ppm 6 – 8 ppm
  31. 31. Animal health ● Check for common symptoms twice a week ● Check for lordosis, as it could indicate P. neurophilia
  32. 32. Handling ● A single netting stressor (2 min) induces changes in cortisol and behavior ● Animals should be caught with a net or a container; consistency in handling is needed – Best way to avoid biases in handling is blinding ● Lighting can be an important factor; high light levels increase anxiety – Always measure light with a calibrated photometer
  33. 33. Issue 2: Anesthesia and drug administration ● While MS-222 is widely used in zebrafish, rapid cooling induces faster and more prolonged anesthesia with less lethality (Chen et al., 2014) ● During induction, spontaneous ventilation (e.g. gill movement) should be monitored closely and can be used as an indicator of the depth of anesthesia. ● Immediately after anesthesia, animals should be transferred to a “surgical table” – Soft sponge with a cut in the middle, which will hold the fish for injection – Sponge should be put inside a container with water; the wet sponge will keep gill perfusion ● Injection should be made with a microsyringe
  34. 34. Intraperitoneal vs. waterborne drug administration ● Waterborne administration is the primary method in the literature – Drug (presumably) absorbed by gills and mouth – Easier to compare efficacy across zebrafish experiments – Less invasive; more suitable for chronic treatment – Many neural receptors are also found in the gills! – MAIN DISADVANTAGE: INTRODUCTION OF PERFORMANCE BIAS AND PSEUDOREPLICATION ● I.P. is much less used – Easier to compare efficacy across species – Reduces performance bias; only alternative for more expensive drugs – More invasive; less suitable for chronic treatment
  35. 35. Issue 3: Pseudoreplication and experimental design ● Pseudo-replication can be introduced whenever animals are treated as a “batch” – Stressor applied to more than 1 animal at each time – Drugs administration – … – Pseudo-replication introduces a confounding variable that cannot be disentangled by statistical analysis ● Unless there is good justification, experiments should always avoid crossover designs, and should ALWAYS account for: – Blinding (of caregivers, experimenters, data analysts) – Randomization ● Good guidelines: ARRIVE, SYRCLE’s RoB