Advertisement

Argus: An open-source and flexible software application for automated quantification of behavior during social interaction in adult zebrafish

  • Soaleha Shams
  • Shahid Amlani
  • Matthew Scicluna
  • Robert Gerlai
Article

Abstract

Zebrafish show great potential for behavioral neuroscience. Promising lines of research, however, require the development and validation of software tools that will allow automated and cost-effective behavioral analysis. Building on our previous work with the RealFishTracker (in-house-developed tracking system), we present Argus, a data extraction and analysis tool built in the open-source R language for behavioral researchers without any expertise in R. Argus includes a new, user-friendly, and efficient graphical user interface, instead of a command-line interface, and offers simplicity and flexibility in measuring complex zebrafish behavior through customizable parameters. In this article, we compare Argus with Noldus EthoVision and Noldus The Observer, to validate this new system. All three software applications were originally designed to quantify the behavior of a single subject. We first also performed an analysis of the movement of individual fish and compared the performance of the three software applications. Next we computed and quantified the behavioral variables that characterize dyadic interactions between zebrafish. We found that Argus and EthoVision extract similar absolute values and patterns of changes in these values for several behavioral measures, including speed, freezing, erratic movement, and interindividual distance. In contrast, the manual coding of behavior in The Observer showed weaker correlations with the two tracking methods (EthoVision and Argus). Thus, Argus is a novel, cost-effective, and customizable method for the analysis of adult zebrafish behavior that may be utilized for the behavioral quantification of both single and dyadic interacting subjects, but further sophistication will be needed for the proper identification of complex motor patterns, measures that a human observers can easily detect.

Keywords

Zebrafish Social behavior Anxiety Dyads R programming language 

Supplementary material

13428_2018_1083_MOESM1_ESM.pdf (959 kb)
ESM 1 (PDF 959 kb)

References

  1. Blaser, R., & Gerlai, R. (2006). Behavioral phenotyping in zebrafish: Comparison of three behavioral quantification methods. Behavior Research Methods, 38, 456–469. doi: https://doi.org/10.3758/BF03192800 CrossRefPubMedGoogle Scholar
  2. Blaser, R. E., Chadwick, L., & McGinnis, G. C. (2010). Behavioral measures of anxiety in zebrafish (Danio rerio). Behavioural Brain Research, 208, 56–62. doi: https://doi.org/10.1016/j.bbr.2009.11.009 CrossRefPubMedGoogle Scholar
  3. Breacker, C., Barber, I., Norton, W. H., McDearmid, J. R., & Tilley, C. A. (2017). A low-cost method of skin swabbing for the collection of DNA samples from small laboratory fish. Zebrafish, 14, 35–41. doi: https://doi.org/10.1089/zeb.2016.1348 CrossRefPubMedPubMedCentralGoogle Scholar
  4. Buske, C., & Gerlai, R. (2011). Shoaling develops with age in Zebrafish (Danio rerio). Prog Neuropsychopharmacol Biol Psychiatry, 35(6), 1409–1415.  https://doi.org/10.1016/j.pnpbp.2010.09.003
  5. Buske, C., & Gerlai, R. (2012). Maturation of shoaling behavior is accompanied by changes in the dopaminergic and serotoninergic systems in zebrafish. Dev Psychobiol, 54(1), 28–35.  https://doi.org/10.1002/dev.20571
  6. Buske, C., & Gerlai, R. (2014). Diving deeper into Zebrafish development of social behavior: Analyzing high resolution data. Journal of Neuroscience Methods, 234, 66–72. doi: https://doi.org/10.1016/j.jneumeth.2014.06.019 CrossRefPubMedGoogle Scholar
  7. Carter, B. S., Cortes-Campos, C., Chen, X., McCammon, J. M., & Sive, H. L. (2017). Validation of protein knockout in mutant zebrafish lines using in vitro translation assays. Zebrafish, 14, 73–76. doi: https://doi.org/10.1089/zeb.2016.1326 CrossRefPubMedGoogle Scholar
  8. Creton, R. (2009). Automated analysis of behavior in zebrafish larvae. Behavioural Brain Research, 203, 127–136. doi: https://doi.org/10.1016/j.bbr.2009.04.030 CrossRefPubMedGoogle Scholar
  9. Estepa, A., & Coll, J. (2015). Innate multigene family memories are implicated in the viral-survivor zebrafish phenotype. PLoS ONE, 10, e0135483. doi: https://doi.org/10.1371/journal.pone.0135483 CrossRefPubMedPubMedCentralGoogle Scholar
  10. Felix, L. M., Antunes, L. M., Coimbra, A. M., & Valentim, A. M. (2017). Behavioral alterations of zebrafish larvae after early embryonic exposure to ketamine. Psychopharmacology, 234, 549–558. doi: https://doi.org/10.1007/s00213-016-4491-7
  11. Field, H. A., Kelley, K. A., Martell, L., Goldstein, A. M., & Serluca, F. C. (2009). Analysis of gastrointestinal physiology using a novel intestinal transit assay in zebrafish. Neurogastroenterology & Motility, 21, 304–312. doi: https://doi.org/10.1111/j.1365-2982.2008.01234.x CrossRefGoogle Scholar
  12. Gerlai, R. (2010). High-throughput behavioral screens: The first step toward finding genes involved in vertebrate brain function using zebrafish. Molecules, 15, 2609–2622. doi: https://doi.org/10.3390/molecules15042609 CrossRefPubMedGoogle Scholar
  13. Gerlai, R. (2012). Using zebrafish to unravel the genetics of complex brain disorders. Current Topics in Behavioral Neuroscience, 12, 3–24. doi: https://doi.org/10.1007/7854_2011_180 CrossRefGoogle Scholar
  14. Gerlai, R. (2014). Fish in behavior research: Unique tools with a great promise! Journal of Neuroscience Methods, 234, 54–58. doi: https://doi.org/10.1016/j.jneumeth.2014.04.015 CrossRefPubMedGoogle Scholar
  15. Gerlai, R. (2015). Zebrafish phenomics: behavioral screens and phenotyping of mutagenized fish. Current Opinions in Behavioral Science, 2, 21–27. doi: https://doi.org/10.1016/j.cobeha.2014.07.007 CrossRefGoogle Scholar
  16. Guo, S., Wagle, M., & Mathur, P. (2012). Toward molecular genetic dissection of neural circuits for emotional and motivational behaviors. Developmental Neurobiology, 72, 358–365. doi: https://doi.org/10.1002/dneu.20927 CrossRefPubMedGoogle Scholar
  17. Jhuang, H., Garrote, E., Mutch, J., Yu, X., Khilnani, V., Poggio, T., … Serre, T. (2010). Automated home-cage behavioural phenotyping of mice. Nature Communications, 1, 68. doi: https://doi.org/10.1038/ncomms1064 CrossRefPubMedGoogle Scholar
  18. Kalueff, A. V., Gebhardt, M., Stewart, A. M., Cachat, J. M., Brimmer, M., Chawla, J. S., … Schneider, H. (2013). Toward a comprehensive catalog of zebrafish behavior 1.0 and beyond. Zebrafish, 10, 70–86. doi: https://doi.org/10.1089/zeb.2012.0861 CrossRefPubMedPubMedCentralGoogle Scholar
  19. Kalueff, A. V., Stewart, A. M., & Gerlai, R. (2014). Zebrafish as an emerging model for studying complex brain disorders. Trends in Pharmacological Sciences, 35, 63–75. doi: https://doi.org/10.1016/j.tips.2013.12.002 CrossRefPubMedPubMedCentralGoogle Scholar
  20. Khan, K. M., Collier, A. D., Meshalkina, D. A., Kysil, E. V., Khatsko, S. L., Kolesnikova, T., … Echevarria, D. J. (2017). Zebrafish models in neuropsychopharmacology and CNS drug discovery. British Journal of Pharmacology, 174, 1925–1944. doi: https://doi.org/10.1111/bph.13754 CrossRefPubMedPubMedCentralGoogle Scholar
  21. Ladu, F., Butail, S., Macri, S., & Porfiri, M. (2014). Sociality modulates the effects of ethanol in zebra fish. Alcoholism: Clinical and Experimental Research, 38, 2096–2104. doi: https://doi.org/10.1111/acer.12432 CrossRefGoogle Scholar
  22. Lawson, N. D. (2016). Reverse genetics in zebrafish: Mutants, morphants, and moving forward. Trends in Cellular Biology, 26, 77–79. doi: https://doi.org/10.1016/j.tcb.2015.11.005 CrossRefGoogle Scholar
  23. Lin, E., Craig, C., Lamothe, M., Sarunic, M. V., Beg, M. F., & Tibbits, G. F. (2015). Construction and use of a zebrafish heart voltage and calcium optical mapping system, with integrated electrocardiogram and programmable electrical stimulation. American Journal of Physiology: Regulatory, Integrative and Comparative Physiology, 308, R755–R768. doi: https://doi.org/10.1152/ajpregu.00001.2015 PubMedGoogle Scholar
  24. Mahabir, S., Chatterjee, D., Buske, C., & Gerlai, R. (2013). Maturation of shoaling in two zebrafish strains: A behavioral and neurochemical analysis. Behavioural Brain Research, 247, 1–8. doi: https://doi.org/10.1016/j.bbr.2013.03.013 CrossRefPubMedPubMedCentralGoogle Scholar
  25. Makhankov, Y. V., Rinner, O., & Neuhauss, S. C. (2004). An inexpensive device for non-invasive electroretinography in small aquatic vertebrates. Journal of Neuroscience Methods, 135, 205–210. doi: https://doi.org/10.1016/j.jneumeth.2003.12.015 CrossRefPubMedGoogle Scholar
  26. Mathur, P., & Guo, S. (2010). Use of zebrafish as a model to understand mechanisms of addiction and complex neurobehavioral phenotypes. Neurobiology of Disease, 40, 66–72. doi: https://doi.org/10.1016/j.nbd.2010.05.016 CrossRefPubMedPubMedCentralGoogle Scholar
  27. Meshalkina, D. A., Kizlyk, M. N., Kysil, E. V., Collier, A. D., Echevarria, D. J., Abreu, M. S., … Kalueff, A. V. (2018). Zebrafish models of autism spectrum disorder. Experimental Neurology, 299, 207–216. doi: https://doi.org/10.1016/j.expneurol.2017.02.004 CrossRefPubMedGoogle Scholar
  28. Miller, N., & Gerlai, R. (2007). Quantification of shoaling behaviour in zebrafish (Danio rerio). Behavioural Brain Research, 184, 157–166. doi: https://doi.org/10.1016/j.bbr.2007.07.007 CrossRefPubMedGoogle Scholar
  29. Miller, N., & Gerlai, R. (2012). From schooling to shoaling: Patterns of collective motion in zebrafish (Danio rerio). PloS ONE, 7, e48865. doi: https://doi.org/10.1371/journal.pone.0048865 CrossRefPubMedPubMedCentralGoogle Scholar
  30. Miller, N. Y., & Gerlai, R. (2011). Shoaling in zebrafish: What we don’t know. Reviews in the Neurosciences, 22, 17–25. doi: https://doi.org/10.1515/rns.2011.004 CrossRefPubMedGoogle Scholar
  31. Moretz, J. A., Martins, E. P., & Robison, B. D. (2007). The effects of early and adult social environment on zebrafish (Danio rerio) behavior. Environmental Biology of Fishes, 80, 91–101. doi: https://doi.org/10.1007/s10641-006-9122-4 CrossRefGoogle Scholar
  32. Mwaffo, V., Butail, S., di Bernardo, M., & Porfiri, M. (2015). Measuring zebrafish turning rate. Zebrafish, 12, 250–254. doi: https://doi.org/10.1089/zeb.2015.1081 CrossRefPubMedGoogle Scholar
  33. Nema, S., Hasan, W., Bhargava, A., & Bhargava, Y. (2016). A novel method for automated tracking and quantification of adult zebrafish behaviour during anxiety. Journal of Neuroscience Methods, 271, 65–75. doi: https://doi.org/10.1016/j.jneumeth.2016.07.004 CrossRefPubMedGoogle Scholar
  34. Nilsen, B. M., Berg, K., Eidem, J. K., Kristiansen, S. I., Brion, F., Porcher, J. M., & Goksoyr, A. (2004). Development of quantitative vitellogenin-ELISAs for fish test species used in endocrine disruptor screening. Analytical and Bioanalytical Chemistry, 378, 621–633. doi: https://doi.org/10.1007/s00216-003-2241-2 CrossRefPubMedGoogle Scholar
  35. Noldus, L. P. J. J., Trienes, R. J. H., Hendriksen, A. H. M., Jansen, H., & Jansen, R. G. (2000). The Observer Video-Pro: New software for the collection, management, and presentation of time-structured data from videotapes and digital media files. Behavior Research Methods, Instruments, & Computers, 32, 197–206. doi: https://doi.org/10.3758/BF03200802 CrossRefGoogle Scholar
  36. Norton, W. (2013). Toward developmental models of psychiatric disorders in zebrafish. Frontiers in Neural Circuits, 7, 79. doi: https://doi.org/10.3389/fncir.2013.00079 CrossRefPubMedPubMedCentralGoogle Scholar
  37. Pagnussat, N., Piato, A. L., Schaefer, I. C., Blank, M., Tamborski, A. R., Guerim, L. D., … Lara, D. R. (2013). One for all and all for one: The importance of shoaling on behavioral and stress responses in zebrafish. Zebrafish, 10, 338–342. doi: https://doi.org/10.1089/zeb.2013.0867 CrossRefPubMedGoogle Scholar
  38. Pelkowski, S. D., Kapoor, M., Richendrfer, H. A., Wang, X., Colwill, R. M., & Creton, R. (2011). A novel high-throughput imaging system for automated analyses of avoidance behavior in zebrafish larvae. Behavioural Brain Research, 223, 135–144. doi: https://doi.org/10.1016/j.bbr.2011.04.033 CrossRefPubMedPubMedCentralGoogle Scholar
  39. Prykhozhij, S. V., Steele, S. L., Razaghi, B., & Berman, J. N. (2017). A rapid and effective method for screening, sequencing and reporter verification of engineered frameshift mutations in zebrafish. Disease Models & Mechanisms, 10, 811–822. doi: https://doi.org/10.1242/dmm.026765 CrossRefGoogle Scholar
  40. Qin, M., Wong, A., Seguin, D., & Gerlai, R. (2014). Induction of social behavior in zebrafish: live versus computer animated fish as stimuli. Zebrafish, 11, 185–197. doi: https://doi.org/10.1089/zeb.2013.0969 CrossRefPubMedPubMedCentralGoogle Scholar
  41. Saif, M., Chatterjee, D., Buske, C., & Gerlai, R. (2013). Sight of conspecific images induces changes in neurochemistry in zebrafish. Behavioural Brain Research, 243, 294–299. doi: https://doi.org/10.1016/j.bbr.2013.01.020 CrossRefPubMedPubMedCentralGoogle Scholar
  42. Saverino, C., & Gerlai, R. (2008). The social zebrafish: Behavioral responses to conspecific, heterospecific, and computer animated fish. Behavioural Brain Research, 191, 77–87. doi: https://doi.org/10.1016/j.bbr.2008.03.013 CrossRefPubMedPubMedCentralGoogle Scholar
  43. Schaefer, I. C., Siebel, A. M., Piato, A. L., Bonan, C. D., Vianna, M. R., & Lara, D. R. (2015). The side-by-side exploratory test: a simple automated protocol for the evaluation of adult zebrafish behavior simultaneously with social interaction. Behavioural Pharmacology, 26, 691–696. doi: https://doi.org/10.1097/fbp.0000000000000145 CrossRefPubMedGoogle Scholar
  44. Schroeder, P., Jones, S., Young, I. S., & Sneddon, L. U. (2014). What do zebrafish want? Impact of social grouping, dominance and gender on preference for enrichment. Lab Animal, 48, 328–337. doi: https://doi.org/10.1177/0023677214538239 CrossRefGoogle Scholar
  45. Seguin, D., & Gerlai, R. (2017). Zebrafish prefer larger to smaller shoals: Analysis of quantity estimation in a genetically tractable model organism. Animal Cognition, 20, 813–821. doi: https://doi.org/10.1007/s10071-017-1102-x CrossRefPubMedGoogle Scholar
  46. Seguin, D. (2018). Effects of early embryonic ethanol exposure on adult zebrafish social behavior. Toronto: PhD, University of TorontoGoogle Scholar
  47. Shams, S., Amlani, S., Buske, C., Chatterjee, D., & Gerlai, R. (2018). Developmental social isolation affects adult behavior, social interaction, and dopamine metabolite levels in zebrafish. Developmental Psychobiology, 60, 43–56. doi:10.1002/dev.21581Google Scholar
  48. Shams, S., & Gerlai, R. (2016). Pharmacological manipulation of shoaling behavior in zebrafish. Current Psychopharmacology, 5, 180–193. doi: https://doi.org/10.2174/2211556005666160607094906 CrossRefGoogle Scholar
  49. Sison, M., Cawker, J., Buske, C., & Gerlai, R. (2006). Fishing for genes influencing vertebrate behavior: Zebrafish making headway. Lab Animal, 35, 33–39. doi: https://doi.org/10.1038/laban0506-33 CrossRefPubMedGoogle Scholar
  50. Teles, M. C., Dahlbom, S. J., Winberg, S., & Oliveira, R. F. (2013). Social modulation of brain monoamine levels in zebrafish. Behavioural Brain Research, 253, 17–24. doi: https://doi.org/10.1016/j.bbr.2013.07.012 CrossRefPubMedGoogle Scholar
  51. Teles, M. C., & Oliveira, R. F. (2016). Quantifying aggressive behavior in zebrafish. Methods in Molecular Biology, 1451, 293–305. doi: https://doi.org/10.1007/978-1-4939-3771-4_20 CrossRefPubMedGoogle Scholar
  52. Thorn, R. J., Clift, D. E., Ojo, O., Colwill, R. M., & Creton, R. (2017). The loss and recovery of vertebrate vision examined in microplates. PloS ONE, 12, e0183414. doi: https://doi.org/10.1371/journal.pone.0183414 CrossRefPubMedPubMedCentralGoogle Scholar
  53. Wang, J., Zhang, X., Shan, R., Ma, S., Tian, H., Wang, W., & Ru, S. (2016). Lipovitellin as an antigen to improve the precision of sandwich ELISA for quantifying zebrafish (Danio rerio) vitellogenin. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 185–186, 87–93. doi: https://doi.org/10.1016/j.cbpc.2016.03.007 Google Scholar
  54. Wright, D., Ward, A. J., Croft, D. P., & Krause, J. (2006). Social organization, grouping, and domestication in fish. Zebrafish, 3, 141–155. doi: https://doi.org/10.1089/zeb.2006.3.141 CrossRefPubMedGoogle Scholar

Copyright information

© Psychonomic Society, Inc. 2018

Authors and Affiliations

  1. 1.Department of Cell & Systems BiologyUniversity of Toronto MississaugaMississaugaCanada
  2. 2.Department of PsychologyUniversity of Toronto MississaugaMississaugaCanada
  3. 3.Department of Computer ScienceUniversity of MontrealMontrealCanada

Personalised recommendations