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Assessment of a novel color vision optomotor response assay in zebrafish larvae with red cone ablation

Lab Animal volume 54, pages 200–206 (2025)Cite this article

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Abstract

Zebrafish are widely used to investigate visual function, owing to their well-defined retinal organization and optical transparency during early developmental stages. Here, we evaluate the effectiveness of a color vision optomotor response (CV-OMR) assay for assessing zebrafish with red cone ablation. Tg (thrb:gal4;UAS:epNTR-p2a-mCherry) transgenic zebrafish were used to ablate red cones via the addition of metronidazole (MTZ). Transgenic zebrafish larvae were treated with MTZ for 0, 12 and 24 h at 5 days post-fertilization, resulting in Tg(+)MTZ(−), Tg(+)MTZ(+)12 h and Tg(+)MTZ(+)24 h groups, respectively. The areas of mCherry-expressing cells, representing red cones in tissue sections, were compared. The mean mCherry expression area was smallest in the Tg(+)MTZ(+)24 h group (16.5 ± 7.6 μm2), followed by the Tg(+)MTZ(+)12 h (404.1 ± 200.9 μm2) and Tg(+)MTZ(−) groups (1,066.6 ± 252.2 μm2; P < 0.001). At 6 days post-fertilization, zebrafish larvae were evaluated using the CV-OMR assay comprising two colors. Results were reported as the area under the curve of the ratio of larvae at the starting point curve. The ratio of larvae at the starting point decreased most rapidly in the Tg(+)MTZ(−) group and most slowly in the Tg(+)MTZ(+)24 h group. There were significant differences in the area under the curve of the ratio of larvae at the starting point curve among the three groups. In conclusion, the CV-OMR assay demonstrated the ability to differentiate color vision impairment based on the extent of red cone ablation. The CV-OMR assay used in this study may prove valuable for evaluating color vision in zebrafish with various eye diseases.

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Fig. 1: Comparison of immunofluorescence staining and mCherry expression areas among the three experimental groups: Tg(+)MTZ(−), Tg(+)MTZ(+)12 h and Tg(+)MTZ(+)24 h.
Fig. 2: Comparison of the ratio of larvae at the starting point of the response curve in the C-OMR assay after red cone ablation at 5 dpf among four groups: control, Tg(+)MTZ(−), Tg(+)MTZ(+)12 h and Tg(+)MTZ(+)24 h.
Fig. 3: Comparison of the AUC representing the ratio of larvae at the starting point as determined by the C-OMR assay after red cone ablation at 5 dpf among four groups: control, Tg(+)MTZ(−), Tg(+)MTZ(+)12 h and Tg(+)MTZ(+)24 h.
Fig. 4: Comparison of the ratio of larvae at the starting point of the response curve in the CV-OMR assay using Red1, Red2 and Red3 stripes after red cone ablation at 5 dpf among four groups: control, Tg(+)MTZ(−), Tg(+)MTZ(+)12 h and Tg(+)MTZ(+)24 h.
Fig. 5: Comparison of the AUC representing the ratio of larvae at the starting point as determined by the CV-OMR assay using Red1, Red2 and Red3 stripes after red cone ablation at 5 dpf among four groups: control, Tg(+)MTZ(−), Tg(+)MTZ(+)12 h and Tg(+)MTZ(+)24 h.
Fig. 6: Comparison of red color preference percentage among four groups: control, Tg(+)MTZ(−), Tg(+)MTZ(+)12 h and Tg(+)MTZ(+)24 h.

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The data that support the findings of this study are available from the corresponding author upon request. Source data are provided with this paper.

References

  1. Thoreson, W. B. & Dacey, D. M. Diverse cell types, circuits, and mechanisms for color vision in the vertebrate retina. Physiol. Rev. 99, 1527–1573 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Deveau, C. et al. Thyroid hormone receptor beta mutations alter photoreceptor development and function in Danio rerio (zebrafish). PLoS Genet. 16, e1008869 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Lucas, P. W. et al. Evolution and function of routine trichromatic vision in primates. Evolution 57, 2636–2643 (2003).

    PubMed  Google Scholar 

  4. Surridge, A. K., Osorio, D. & Mundy, N. I. Evolution and selection of trichromatic vision in primates. Trends Ecol. Evol. 18, 198–205 (2003).

    Article  Google Scholar 

  5. de Moraes, P. Z., Diniz, P., Spyrides, M. H. C. & Pessoa, D. M. A. The effect of pelage, background, and distance on predator detection and the evolution of primate color vision. Am. J. Primatol. 83, e23230 (2021).

    Article  PubMed  Google Scholar 

  6. Neitz, J. & Neitz, M. The genetics of normal and defective color vision. Vis. Res. 51, 633–651 (2011).

    Article  CAS  PubMed  Google Scholar 

  7. Semenov, E. P. et al. Reading performance in blue cone monochromacy: defining an outcome measure for a clinical trial. Transl. Vis. Sci. Technol. 9, 13 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Garcia-Martin, E. et al. Assessment of visual function and the neuroretina in subjects diagnosed with congenital anomaly of color vision. Opt. Express 31, 5625–5639 (2023).

    Article  PubMed  Google Scholar 

  9. Mancuso, K. et al. Gene therapy for red-green colour blindness in adult primates. Nature 461, 784–787 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Neitz, M. & Neitz, J. Curing color blindness—mice and nonhuman primates. Cold Spring Harb. Perspect. Med. 4, a017418 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Shoji, T., Sato, H., Chihara, E. & Sakurai, Y. Are middle-age blood pressure levels related to color vision impairment? The Okubo Color Study. Am. J. Hypertens. 28, 98–105 (2015).

    Article  CAS  PubMed  Google Scholar 

  12. Dall’Antonia, I., Šonka, K. & Dušek, P. Olfaction and colour vision: what can they tell us about Parkinson’s disease? Prague Med. Rep. 119, 85–96 (2018).

    Article  PubMed  Google Scholar 

  13. Garcia-Martin, E. et al. Visual function and retinal changes in patients with bipolar disorder. Retina 39, 2012–2021 (2019).

    Article  CAS  PubMed  Google Scholar 

  14. Piro, A. et al. Impairment of acquired color vision in multiple sclerosis: an early diagnostic sign linked to the greatness of disease. Int. Ophthalmol. 39, 671–676 (2019).

    Article  PubMed  Google Scholar 

  15. Chen, H. et al. New evidence of central nervous system damage in diabetes: impairment of fine visual discrimination. Diabetes 71, 1772–1784 (2022).

    Article  CAS  PubMed  Google Scholar 

  16. Vit, J. P. et al. Color and contrast vision in mouse models of aging and Alzheimer’s disease using a novel visual-stimuli four-arm maze. Sci. Rep. 11, 1255 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Koida, K. et al. Color vision test for dichromatic and trichromatic macaque monkeys. J. Vis. 13, 1 (2013).

    Article  PubMed  Google Scholar 

  18. Kelber, A., Vorobyev, M. & Osorio, D. Animal colour vision—behavioural tests and physiological concepts. Biol. Rev. Camb. Philos. Soc. 78, 81–118 (2003).

    Article  PubMed  Google Scholar 

  19. Escobar-Camacho, D., Marshall, J. & Carleton, K. L. Behavioral color vision in a cichlid fish: Metriaclima benetos. J. Exp. Biol. 220, 2887–2899 (2017).

    PubMed  PubMed Central  Google Scholar 

  20. Howe, K. et al. The zebrafish reference genome sequence and its relationship to the human genome. Nature 496, 498–503 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Chhetri, J., Jacobson, G. & Gueven, N. Zebrafish—on the move towards ophthalmological research. Eye 28, 367–380 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Bilotta, J. & Saszik, S. The zebrafish as a model visual system. Int. J. Dev. Neurosci. 19, 621–629 (2001).

    Article  CAS  PubMed  Google Scholar 

  23. Meier, A., Nelson, R. & Connaughton, V. P. Color processing in zebrafish retina. Front. Cell Neurosci. 12, 327 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Mackin, R. D. et al. Endocrine regulation of multichromatic color vision. Proc. Natl Acad. Sci. USA 116, 16882–16891 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Fornetto, C., Tiso, N., Pavone, F. S. & Vanzi, F. Colored visual stimuli evoke spectrally tuned neuronal responses across the central nervous system of zebrafish larvae. BMC Biol. 18, 172 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Robinson, J., Schmitt, E. A., Hárosi, F. I., Reece, R. J. & Dowling, J. E. Zebrafish ultraviolet visual pigment: absorption spectrum, sequence, and localization. Proc. Natl Acad. Sci. USA 90, 6009–6012 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Maaswinkel, H. & Li, L. Spatio-temporal frequency characteristics of the optomotor response in zebrafish. Vision Res. 43, 21–30 (2003).

    Article  PubMed  Google Scholar 

  28. Koun, S. et al. Development of an experimental model for ocular toxicity screening in zebrafish. Biochem. Biophys. Res. Commun. 559, 155–160 (2021).

    Article  CAS  PubMed  Google Scholar 

  29. Hagerman, G. F. et al. Rapid recovery of visual function associated with blue cone ablation in zebrafish. PLoS ONE 11, e0166932 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  30. Kaiser, W. The spectral sensitivity of the honeybee’s optomotor walking response. J. Comp. Physiol. 90, 405–408 (1974).

    Article  Google Scholar 

  31. Schaerer, S. & Neumeyer, C. Motion detection in goldfish investigated with the optomotor response is ‘color blind’. Vis. Res. 36, 4025–4034 (1996).

    Article  CAS  PubMed  Google Scholar 

  32. Orger, M. B. & Baier, H. Channeling of red and green cone inputs to the zebrafish optomotor response. Vis. Neurosci. 22, 275–281 (2005).

    Article  PubMed  Google Scholar 

  33. Nelson, R. F., Balraj, A., Suresh, T., Torvund, M. & Patterson, S. S. Strain variations in cone wavelength peaks in situ during zebrafish development. Vis. Neurosci. 36, E010 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  34. Dingsdag, S. A. & Hunter, N. Metronidazole: an update on metabolism, structure-cytotoxicity and resistance mechanisms. J. Antimicrob. Chemother. 73, 265–279 (2018).

    Article  CAS  PubMed  Google Scholar 

  35. Curado, S., Stainier, D. Y. & Anderson, R. M. Nitroreductase-mediated cell/tissue ablation in zebrafish: a spatially and temporally controlled ablation method with applications in developmental and regeneration studies. Nat. Protoc. 3, 948–954 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Pisharath, H., Rhee, J. M., Swanson, M. A., Leach, S. D. & Parsons, M. J. Targeted ablation of beta cells in the embryonic zebrafish pancreas using E. coli nitroreductase. Mech. Dev. 124, 218–229 (2007).

    Article  CAS  PubMed  Google Scholar 

  37. Jägle, H., de Luca, E., Serey, L., Bach, M. & Sharpe, L. T. Visual acuity and X-linked color blindness. Graefes Arch. Clin. Exp. Ophthalmol. 244, 447–453 (2006).

    Article  PubMed  Google Scholar 

  38. Thibos, L. N., Ye, M., Zhang, X. & Bradley, A. The chromatic eye: a new reduced-eye model of ocular chromatic aberration in humans. Appl. Opt. 31, 3594–3600 (1992).

    Article  CAS  PubMed  Google Scholar 

  39. Fraser, B., DuVal, M. G., Wang, H. & Allison, W. T. Regeneration of cone photoreceptors when cell ablation is primarily restricted to a particular cone subtype. PLoS ONE 8, e55410 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Vemala, R., Sivaprasad, S. & Barbur, J. L. Detection of early loss of color vision in age-related macular degeneration—with emphasis on drusen and reticular pseudodrusen. Invest. Ophthalmol. Vis. Sci. 58, Bio247–bio254 (2017).

    Article  PubMed  Google Scholar 

  41. Kuehlewein, L. et al. Central visual function and genotype-phenotype correlations in PDE6A-associated retinitis pigmentosa. Invest. Ophthalmol. Vis. Sci. 63, 9 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Li, M. et al. Effects of acute high intraocular pressure on red-green and blue-yellow cortical color responses in non-human primates. NeuroImage Clin. 35, 103092 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  43. Bayer, L., Funk, J. & Töteberg-Harms, M. Incidence of dyschromatopsy in glaucoma. Int. Ophthalmol. 40, 597–605 (2020).

    Article  PubMed  Google Scholar 

  44. Kassen, S. C. et al. Time course analysis of gene expression during light-induced photoreceptor cell death and regeneration in albino zebrafish. Dev. Neurobiol. 67, 1009–1031 (2007).

    Article  CAS  PubMed  Google Scholar 

  45. ARVO statement for the use of animals in ophthalmic and vision research. ARVO https://www.arvo.org/About/policies/statement-for-the-use-of-animals-in-ophthalmic-and-vision-research (2023).

  46. The ARRIVE guidelines (animal research: reporting of in vivo experiments). ARRIVE https://arriveguidelines.org (2023).

  47. Davison, J. M. et al. Transactivation from Gal4-VP16 transgenic insertions for tissue-specific cell labeling and ablation in zebrafish. Dev. Biol. 304, 811–824 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Yoshimatsu, T. et al. Presynaptic partner selection during retinal circuit reassembly varies with timing of neuronal regeneration in vivo. Nat. Commun. 7, 10590 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. D’Orazi, F. D., Suzuki, S. C., Darling, N., Wong, R. O. & Yoshimatsu, T. Conditional and biased regeneration of cone photoreceptor types in the zebrafish retina. J. Comp. Neurol. 528, 2816–2830 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  50. Chung, A. Y. et al. Generation of demyelination models by targeted ablation of oligodendrocytes in the zebrafish CNS. Mol. Cells 36, 82–87 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Tabor, K. M. et al. Direct activation of the Mauthner cell by electric field pulses drives ultrarapid escape responses. J. Neurophysiol. 112, 834–844 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  52. Halpern, M. E. et al. Gal4/UAS transgenic tools and their application to zebrafish. Zebrafish 5, 97–110 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Choi, E. K., Choi, B. M., Cho, Y. & Kim, S. Myelin toxicity of chlorhexidine in zebrafish larvae. Pediatr. Res. https://doi.org/10.1038/s41390-022-02186-6 (2022).

  54. Eom, Y. et al. Effect of titanium dioxide nanoparticle exposure on the ocular surface: an animal study. Ocul. Surf. 14, 224–232 (2016).

    Article  PubMed  Google Scholar 

  55. Fliotsos, M. J., Zhao, J., Pradeep, T., Ighani, M. & Eghrari, A. O. Testing a popular smartphone application for colour vision assessment in healthy volunteer subjects. Neuroophthalmology 45, 99–104 (2021).

    Article  PubMed  Google Scholar 

  56. Zhao, J., Fliotsos, M. J., Ighani, M. & Eghrari, A. O. Comparison of a smartphone application with Ishihara pseudoisochromatic plate for testing colour vision. Neuroophthalmology 43, 235–239 (2019).

    Article  PubMed  Google Scholar 

  57. Coblis-Color Blindness Simulator. Colblindor https://www.color-blindness.com/coblis-color-blindness-simulator (2023).

Download references

Acknowledgements

We express our gratitude to R. O. Wong (University of Washington) and T. Yoshimatsu for providing the pCG2thrb:gal4 DNA plasmid, as well as I. Jeong for the UAS:epNTR-p2a-mCherry plasmid used in this study. This work was supported by the SNUBH Research Fund (grant number 13-2020-007), by a TRC Research Grant of the Korea University Medicine and Korea Institute of Science and Technology (K2107901), by Korea University Ansan Hospital grant, by Korea University grants (K1625491, K1722121, K1811051, K1913161 and K2010921), by the Korea Medical Device Development Fund grant funded by the Korea government (the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy, the Ministry of Health & Welfare, the Ministry of Food and Drug Safety) (project number 1711174253, RS-2020-KD000296), by the Korea Environment Industry & Technology Institute (KEITI) through the Technology Development Project for Safety Management of Household Chemical Products, funded by the Korea Ministry of Environment (MOE) (2020002960007, NTIS-1485017544), by the Technology Development Program (S3127902) funded by the Ministry of SMEs and Startups (MSS, Korea), by the Technology Development Program (S3305836) funded by the Ministry of SMEs and Startups (MSS, Korea), by the National Research Foundation of Korea (NRF) grant funded by the Korea Government (MSIT) (NRF-2021R1F1A1062017), and by ‘Technical start-up corporation fostering project’ through the Commercialization Promotion Agency for R&D Outcomes (COMPA) grant funded by the Korea Government (MSIT) (no. RS-2023-00259877). This study was funded in part by a Research to Prevent Blindness Challenge Grant to the Ophthalmology Department at Emory University; National Institutes of Health grants R01EY028450, R01EY021592, R01EY028859 and P30EY006360; The Abraham J. and Phyllis Katz Foundation; and VA RR&D grants I01RX002806, I21RX001924 and VA RR&D C9246C (Atlanta Veterans Administration Center for Excellence in Vision and Neurocognitive Rehabilitation).

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Authors and Affiliations

  1. Department of Ophthalmology, Korea University Ansan Hospital, Ansan-si, Republic of Korea

    Youngsub Eom & Hye-Si Park

  2. Department of Ophthalmology, Korea University College of Medicine, Seoul, Republic of Korea

    Youngsub Eom & Jong Suk Song

  3. Department of Ophthalmology, Emory University School of Medicine, Atlanta, GA, USA

    Youngsub Eom, Jeffrey H. Boatright & John M. Nickerson

  4. Zebrafish Translational Medical Research Center, Korea University, Ansan-si, Republic of Korea

    Youngsub Eom, Hye-Si Park, Hae-Chul Park & Suhyun Kim

  5. Medical College of Georgia, Augusta University, Augusta, GA, USA

    Eunheh Koh

  6. Department of Biomedical Sciences, Korea University College of Medicine, Seoul, Republic of Korea

    Hae-Chul Park & Suhyun Kim

  7. Atlanta VA Center for Visual and Neurocognitive Rehabilitation, Decatur, GA, USA

    Jeffrey H. Boatright

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Contributions

Y.E. conceived and designed the study, acquired data, analyzed and interpreted the data, drafted the manuscript and reviewed the final version. H.-S.P. and S.K. contributed to data acquisition, data analysis and interpretation, and reviewed the final manuscript. E.K. contributed to data interpretation and reviewed the final manuscript. H.-C.P. provided experimental equipment and reviewed the final manuscript. J.S.S., J.H.B. and J.M.N. supervised the study, contributed to data interpretation and reviewed the final manuscript.

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Correspondence to Youngsub Eom or Suhyun Kim.

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Eom, Y., Koh, E., Park, HS. et al. Assessment of a novel color vision optomotor response assay in zebrafish larvae with red cone ablation. Lab Anim 54, 200–206 (2025). https://doi.org/10.1038/s41684-025-01586-5

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