Analyzing the Effects of Age, Time of Day, and Experiment on the Basal Locomotor Activity and Light-Off Visual Motor Response Assays in Zebrafish Larvae
Abstract
:1. Introduction
2. Materials and Methods
2.1. Fish Husbandry and Larvae Production
2.2. Experimental Procedure
2.3. Behavioral Analysis
Behavioral Assessments in Zebrafish Larvae
2.4. Data Analysis
2.5. Statistical Analysis
3. Results
3.1. Basal Locomotor Activity (BLA)
3.2. Light-Off Visual Motor Response (VMR)
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
- Ilie, O.D.; Duta, R.; Jijie, R.; Nita, I.B.; Nicoara, M.; Faggio, C.; Dobrin, R.; Mavroudis, I.; Ciobica, A.; Doroftei, B. Assessing Anti-Social and Aggressive Behavior in a Zebrafish (Danio rerio) Model of Parkinson’s Disease Chronically Exposed to Rotenone. Brain Sci. 2022, 12, 898. [Google Scholar] [CrossRef]
- Rashidian, G.; Mohammadi-Aloucheh, R.; Hosseinzadeh-Otaghvari, F.; Chupani, L.; Stejskal, V.; Samadikhah, H.; Zamanlui, S.; Multisanti, C.R.; Faggio, C. Long-term exposure to small-sized silica nanoparticles (SiO2-NPs) induces oxidative stress and impairs reproductive performance in adult zebrafish (Danio rerio). Comp. Biochem. Physiol. Part C Toxicol. Pharmacol. 2023, 273, 109715. [Google Scholar] [CrossRef] [PubMed]
- Ricarte, M.; Prats, E.; Montemurro, N.; Bedrossiantz, J.; Bellot, M.; Gómez-Canela, C.; Raldúa, D. Environmental concentrations of tire rubber-derived 6PPD-quinone alter CNS function in zebrafish larvae. Sci. Total Environ. 2023, 896, 165240. [Google Scholar] [CrossRef] [PubMed]
- Basnet, R.M.; Zizioli, D.; Taweedet, S.; Finazzi, D.; Memo, M. Zebrafish larvae as a behavioral model in neuropharmacology. Biomedicines 2019, 7, 23. [Google Scholar] [CrossRef] [PubMed]
- Strähle, U.; Scholz, S.; Geisler, R.; Greiner, P.; Hollert, H.; Rastegar, S.; Schumacher, A.; Selderslaghs, I.; Weiss, C.; Witters, H.; et al. Zebrafish embryos as an alternative to animal experiments-A commentary on the definition of the onset of protected life stages in animal welfare regulations. Reprod. Toxicol. 2012, 33, 128–132. [Google Scholar] [CrossRef]
- Hill, B.N.; Coldsnow, K.D.; Hunter, D.L.; Hedge, J.M.; Korest, D.; Jarema, K.A.; Padilla, S. Assessment of Larval Zebrafish Locomotor Activity for Developmental Neurotoxicity Screening. Neuromethods 2021, 172, 327–351. [Google Scholar] [CrossRef]
- Faria, M.; Bellot, M.; Bedrossiantz, J.; Ramírez, J.R.R.; Prats, E.; Garcia-Reyero, N.; Gomez-Canela, C.; Mestres, J.; Rovira, X.; Barata, C.; et al. Environmental levels of carbaryl impair zebrafish larvae behaviour: The potential role of ADRA2B and HTR2B. J. Hazard. Mater. 2022, 431, 128563. [Google Scholar] [CrossRef]
- Faria, M.; Prats, E.; Rosas Ramírez, J.R.; Bellot, M.; Bedrossiantz, J.; Pagano, M.; Valls, A.; Gomez-Canela, C.; Porta, J.M.; Mestres, J.; et al. Androgenic activation, impairment of the monoaminergic system and altered behavior in zebrafish larvae exposed to environmental concentrations of fenitrothion. Sci. Total Environ. 2021, 775, 145671. [Google Scholar] [CrossRef]
- Hill, B.N.; Britton, K.N.; Hunter, D.L.; Olin, J.K.; Lowery, M.; Hedge, J.M.; Knapp, B.R.; Jarema, K.A.; Rowson, Z.; Padilla, S. Inconsistencies in variable reporting and methods in larval zebrafish behavioral assays. Neurotoxicol. Teratol. 2023, 96, 107163. [Google Scholar] [CrossRef]
- Cassar, S.; Adatto, I.; Freeman, J.L.; Gamse, J.T.; Iturria, I.; Lawrence, C.; Muriana, A.; Peterson, R.T.; Van Cruchten, S.; Zon, L.I. Use of Zebrafish in Drug Discovery Toxicology. Chem. Res. Toxicol. 2020, 33, 95–118. [Google Scholar] [CrossRef]
- Rosa, J.G.S.; Lima, C.; Lopes-Ferreira, M. Zebrafish Larvae Behavior Models as a Tool for Drug Screenings and Pre-Clinical Trials: A Review. Int. J. Mol. Sci. 2022, 23, 6647. [Google Scholar] [CrossRef] [PubMed]
- Padilla, S.; Hunter, D.L.; Padnos, B.; Frady, S.; MacPhail, R.C. Assessing locomotor activity in larval zebrafish: Influence of extrinsic and intrinsic variables. Neurotoxicol. Teratol. 2011, 33, 624–630. [Google Scholar] [CrossRef] [PubMed]
- Fero, K.; Yokogawa, T.; Burgess, H.A. The behavioral repertoire of larval zebrafish. In Zebrafish Models in Neurobehavioral Research; Springer: Berlin/Heidelberg, Germany, 2011; pp. 249–291. [Google Scholar]
- Tian, N.; Copenhagen, D.R. Visual stimulation is required for refinement on ON and OFF pathways in postnatal retina. Neuron 2003, 39, 85–96. [Google Scholar] [CrossRef] [PubMed]
- Tufi, S.; Leonards, P.; Lamoree, M.; De Boer, J.; Legler, J.; Legradi, J. Changes in Neurotransmitter Profiles during Early Zebrafish (Danio rerio) Development and after Pesticide Exposure. Environ. Sci. Technol. 2016, 50, 3222–3230. [Google Scholar] [CrossRef] [PubMed]
- Wolter, M.E.; Svoboda, K.R. Doing the locomotion: Insights and potential pitfalls associated with using locomotor activity as a readout of the circadian rhythm in larval zebrafish. J. Neurosci. Methods 2020, 330, 108465. [Google Scholar] [CrossRef] [PubMed]
- Poupard, G.; André, M.; Durliat, M.; Ballagny, C.; Boeuf, G.; Babin, P.J. Apolipoprotein E gene expression correlates with endogenous lipid nutrition and yolk syncytial layer lipoprotein synthesis during fish development. Cell Tissue Res. 2000, 300, 251–261. [Google Scholar] [CrossRef] [PubMed]
- Kimmel, C.B.; Ballard, W.W.; Kimmel, S.R.; Ullmann, B.; Schilling, T.F. Stages of embryonic development of the zebrafish. Dev. Dyn. 1995, 203, 253–310. [Google Scholar] [CrossRef] [PubMed]
- Bedrossiantz, J.; Faria, M.; Prats, E.; Barata, C.; Cachot, J.; Raldúa, D. Heart rate and behavioral responses in three phylogenetically distant aquatic model organisms exposed to environmental concentrations of carbaryl and fenitrothion. Sci. Total Environ. 2023, 865, 161268. [Google Scholar] [CrossRef] [PubMed]
- Cameron, A.; Trivedi, P.K. Microeconometrics Using Stata, 2nd ed.; Stata Press: College Station, TX, USA, 2022; ISBN 978-1-59718-359-8. [Google Scholar]
- Armitage, P. Statistical Methods in Medical Research; John Wiley and Sons: New York, NY, USA, 1971. [Google Scholar]
- StataCorp, L. Stata Statistical Software: Release 17; StataCorp LP: College Station, TX, USA, 2021. [Google Scholar]
- de Esch, C.; van der Linde, H.; Slieker, R.; Willemsen, R.; Wolterbeek, A.; Woutersen, R.; De Groot, D. Locomotor activity assay in zebrafish larvae: Influence of age, strain and ethanol. Neurotoxicol. Teratol. 2012, 34, 425–433. [Google Scholar] [CrossRef]
- Maximino, C.; Meinerz, D.L.; Fontana, B.D.; Mezzomo, N.J.; Stefanello, F.V.; Prestes, A.D.S.; Batista, C.B.; Rubin, M.A.; Barbosa, N.V.; Rocha, J.B.T.; et al. Extending the analysis of zebrafish behavioral endophenotypes for modeling psychiatric disorders: Fear conditioning to conspecific alarm response. Behav. Process. 2018, 149, 35–42. [Google Scholar] [CrossRef]
- Lima, M.G.; do Carmo Silva, R.X.; dos Santos Silva, S.D.N.; dos Santos Rodrigues, L.D.S.; Oliveira, K.R.H.M.; Batista, E.D.J.O.; Maximino, C.; Herculano, A.M. Time-dependent sensitization of stress responses in zebrafish: A putative model for post-traumatic stress disorder. Behav. Process. 2016, 128, 70–82. [Google Scholar] [CrossRef] [PubMed]
- Ingebretson, J.J.; Masino, M.A. Quantification of locomotor activity in larval Zebrafish: Considerations for the design of high-throughput behavioral studies. Front. Neural Circuits 2013, 7, 109. [Google Scholar] [CrossRef] [PubMed]
- Bezzina, L.; Lee, J.C.; Lovibond, P.F.; Colagiuri, B. Extinction and renewal of cue-elicited reward-seeking. Behav. Res. Ther. 2016, 87, 162–169. [Google Scholar] [CrossRef] [PubMed]
- Schepers, S.T.; Bouton, M.E. Hunger as a Context: Food Seeking That Is Inhibited During Hunger Can Renew in the Context of Satiety. Psychol. Sci. 2017, 28, 1640–1648. [Google Scholar] [CrossRef] [PubMed]
- Selderslaghs, I.W.T.; Hooyberghs, J.; De Coen, W.; Witters, H.E. Locomotor activity in zebrafish embryos: A new method to assess developmental neurotoxicity. Neurotoxicol. Teratol. 2010, 32, 460–471. [Google Scholar] [CrossRef] [PubMed]
- Hurd, M.W.; Cahill, G.M. Entraining signals initiate behavioral circadian rhythmicity in larval zebrafish. J. Biol. Rhythms 2002, 17, 307–314. [Google Scholar] [CrossRef]
- MacPhail, R.C.; Brooks, J.; Hunter, D.L.; Padnos, B.; Irons, T.D.; Padilla, S. Locomotion in larval zebrafish: Influence of time of day, lighting and ethanol. Neurotoxicology 2009, 30, 52–58. [Google Scholar] [CrossRef]
- Kristofco, L.A.; Cruz, L.C.; Haddad, S.P.; Behra, M.L.; Chambliss, C.K.; Brooks, B.W. Age matters: Developmental stage of Danio rerio larvae influences photomotor response thresholds to diazinion or diphenhydramine. Aquat. Toxicol. 2016, 170, 344–354. [Google Scholar] [CrossRef] [PubMed]
- Fernandes, A.M.; Fero, K.; Arrenberg, A.B.; Bergeron, S.A.; Driever, W.; Burgess, H.A. Deep brain photoreceptors control light-seeking behavior in zebrafish larvae. Curr. Biol. 2012, 22, 2042–2047. [Google Scholar] [CrossRef]
- Haigis, A.C.; Ottermanns, R.; Schiwy, A.; Hollert, H.; Legradi, J. Getting more out of the zebrafish light dark transition test. Chemosphere 2022, 295, 133863. [Google Scholar] [CrossRef]
- Ogungbemi, A.; Leuthold, D.; Scholz, S.; Küster, E. Hypo- or hyperactivity of zebrafish embryos provoked by neuroactive substances: A review on how experimental parameters impact the predictability of behavior changes. Environ. Sci. Eur. 2019, 31, 88. [Google Scholar] [CrossRef]
- Rock, S.; Rodenburg, F.; Schaaf, M.J.M.; Tudorache, C. Detailed Analysis of Zebrafish Larval Behaviour in the Light Dark Challenge Assay Shows That Diel Hatching Time Determines Individual Variation. Front. Physiol. 2022, 13, 827282. [Google Scholar] [CrossRef] [PubMed]
- Fraser, T.W.K.; Khezri, A.; Jusdado, J.G.H.; Lewandowska-Sabat, A.M.; Henry, T.; Ropstad, E. Toxicant induced behavioural aberrations in larval zebrafish are dependent on minor methodological alterations. Toxicol. Lett. 2017, 276, 62–68. [Google Scholar] [CrossRef] [PubMed]
- Fitzgerald, J.A.; Kirla, K.T.; Zinner, C.P.; vom Berg, C.M. Emergence of consistent intra-individual locomotor patterns during zebrafish development. Sci. Rep. 2019, 9, 13647. [Google Scholar] [CrossRef] [PubMed]
- Nüßer, L.K.; Skulovich, O.; Hartmann, S.; Seiler, T.B.; Cofalla, C.; Schuettrumpf, H.; Hollert, H.; Salomons, E.; Ostfeld, A. A sensitive biomarker for the detection of aquatic contamination based on behavioral assays using zebrafish larvae. Ecotoxicol. Environ. Saf. 2016, 133, 271–280. [Google Scholar] [CrossRef] [PubMed]
- Maeda, H.; Fukushima, N.; Hasumi, A. Standardized method for the assessment of behavioral responses of zebrafish larvae. Biomedicines 2021, 9, 884. [Google Scholar] [CrossRef]
- Faria, M.; Prats, E.; Novoa-Luna, K.A.; Bedrossiantz, J.; Gómez-Canela, C.; Gómez-Oliván, L.M.; Raldúa, D. Development of a vibrational startle response assay for screening environmental pollutants and drugs impairing predator avoidance. Sci. Total Environ. 2019, 650, 87–96. [Google Scholar] [CrossRef]
N | n | SD | SDBetween | SDWithin | Rho | 1-Rho | ||
---|---|---|---|---|---|---|---|---|
3814 | 143 | 133.14 | 28.99 | 11.75 | 26.51 | 16.43% | 83.57% | |
Day (age) | ||||||||
5 | 954 | 143 | 132.38 | 34.42 | 22.36 | 26.17 | 42.19% | 57.81% |
6 | 954 | 143 | 141.15 | 30.20 | 19.28 | 23.55 | 40.13% | 59.87% |
7 | 952 | 143 | 131.09 | 22.99 | 14.10 | 18.21 | 37.48% | 62.52% |
8 | 954 | 143 | 127.91 | 25.31 | 16.37 | 19.31 | 41.82% | 58.18% |
Time (hours) | ||||||||
08:00 | 572 | 143 | 133.81 | 30.30 | 18.07 | 24.36 | 35.51% | 64.49% |
10:00 | 572 | 143 | 137.06 | 27.94 | 16.83 | 22.34 | 36.20% | 63.80% |
12:00 | 572 | 143 | 139.87 | 31.68 | 19.33 | 25.14 | 37.15% | 62.85% |
14:00 | 572 | 143 | 135.18 | 26.78 | 15.03 | 22.19 | 31.43% | 68.57% |
16:00 | 571 | 143 | 140.22 | 28.60 | 17.12 | 22.97 | 35.71% | 64.29% |
18:00 | 571 | 143 | 124.46 | 22.63 | 12.67 | 18.77 | 31.29% | 68.71% |
20:00 | 384 | 96 | 115.58 | 26.32 | 14.38 | 22.09 | 29.76% | 70.24% |
Experiment | ||||||||
1 | 1344 | 48 | 129.17 | 25.77 | 9.89 | 23.84 | 14.67% | 85.33% |
2 | 1342 | 48 | 135.80 | 31.14 | 13.02 | 28.36 | 17.40% | 82.60% |
3 | 1128 | 47 | 134.69 | 29.45 | 11.28 | 27.25 | 14.64% | 85.36% |
N | n | SD | SDBetween | SDWithin | Rho | 1-Rho | ||
---|---|---|---|---|---|---|---|---|
3699 | 143 | 12.79 | 8.56 | 3.65 | 7.76 | 18.14% | 81.86% | |
Day (age) | ||||||||
5 | 951 | 143 | 12.90 | 8.75 | 5.14 | 7.13 | 34.14% | 65.86% |
6 | 950 | 143 | 13.33 | 8.54 | 5.66 | 6.50 | 43.10% | 56.90% |
7 | 900 | 143 | 12.96 | 8.05 | 5.44 | 6.11 | 44.26% | 55.74% |
8 | 898 | 143 | 11.92 | 8.82 | 6.27 | 6.39 | 49.09% | 50.91% |
Time (hours) | ||||||||
08:00 | 473 | 143 | 14.45 | 7.67 | 4.96 | 6.05 | 40.16% | 59.84% |
10:00 | 570 | 143 | 11.28 | 7.46 | 4.42 | 6.02 | 35.04% | 64.96% |
12:00 | 568 | 143 | 11.59 | 8.28 | 4.45 | 6.98 | 28.85% | 71.15% |
14:00 | 567 | 143 | 11.94 | 9.34 | 5.22 | 7.76 | 31.20% | 68.80% |
16:00 | 571 | 143 | 12.13 | 8.61 | 5.15 | 6.94 | 35.51% | 64.49% |
18:00 | 569 | 143 | 14.18 | 9.60 | 5.61 | 7.80 | 34.12% | 65.88% |
20:00 | 381 | 96 | 14.91 | 7.57 | 5.03 | 5.68 | 43.91% | 56.09% |
Experiment | ||||||||
1 | 1244 | 48 | 11.41 | 6.21 | 2.83 | 5.55 | 20.61% | 79.39% |
2 | 1341 | 48 | 13.75 | 7.29 | 3.41 | 6.46 | 21.73% | 78.27% |
3 | 1114 | 47 | 13.17 | 11.53 | 4.26 | 10.74 | 13.57% | 86.43% |
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Tagkalidou, N.; Multisanti, C.R.; Bleda, M.J.; Bedrossiantz, J.; Prats, E.; Faggio, C.; Barata, C.; Raldúa, D. Analyzing the Effects of Age, Time of Day, and Experiment on the Basal Locomotor Activity and Light-Off Visual Motor Response Assays in Zebrafish Larvae. Toxics 2024, 12, 349. https://doi.org/10.3390/toxics12050349
Tagkalidou N, Multisanti CR, Bleda MJ, Bedrossiantz J, Prats E, Faggio C, Barata C, Raldúa D. Analyzing the Effects of Age, Time of Day, and Experiment on the Basal Locomotor Activity and Light-Off Visual Motor Response Assays in Zebrafish Larvae. Toxics. 2024; 12(5):349. https://doi.org/10.3390/toxics12050349
Chicago/Turabian StyleTagkalidou, Niki, Cristiana Roberta Multisanti, Maria Jose Bleda, Juliette Bedrossiantz, Eva Prats, Caterina Faggio, Carlos Barata, and Demetrio Raldúa. 2024. "Analyzing the Effects of Age, Time of Day, and Experiment on the Basal Locomotor Activity and Light-Off Visual Motor Response Assays in Zebrafish Larvae" Toxics 12, no. 5: 349. https://doi.org/10.3390/toxics12050349