Oxytocin protective effects on zebrafish larvae models of autism-like spectrum disorder

Document Type : Original Article


1 Department of Aquatic Animal Health, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

2 Centre of Excellence for Warm Water Fish Health and Disease, Shahid Chamran University of Ahvaz, Ahvaz, Iran

3 Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS), Tehran, Iran

4 Department of Toxicology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

5 Oncopathology Research Center, Iran University of Medical Sciences (IUMS), Tehran, Iran

6 Toxicology and Diseases Group (TDG), Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences (TUMS), Tehran, Iran



Objective(s): Autism is a complicated neurodevelopmental disorder characterized by social interaction deficiencies, hyperactivity, anxiety, communication disorders, and a limited range of interests. The zebrafish (Danio rerio) is a social vertebrate used as a biomedical research model to understand social behavior mechanisms. 
Materials and Methods: After spawning, the eggs were exposed to sodium valproate for 48 hr, after which the eggs were divided into eight groups. Except for the positive and control groups, there were six treatment groups based on oxytocin concentration (25, 50, and 100 μM) and time point (24 and 48 hr). Treatment was performed on days 6 and 7, examined by labeling oxytocin with fluorescein-5-isothiocyanate (FITC) and imaging with confocal microscopy and the expression levels of potential genes associated with the qPCR technique. Behavioral studies, including light-dark background preference test, shoaling behavior, mirror test, and social preference, were performed on 10, 11, 12, and 13 days post fertilization (dpf), respectively.
Results: The results showed that the most significant effect of oxytocin was at the concentration of 50 μM and the time point of 48 hr. Increased expression of shank3a, shank3b, and oxytocin receptor genes was also significant at this oxytocin concentration. Light-dark background preference results showed that oxytocin in the concentration of 50 µM significantly increased the number of crosses between dark and light areas compared with valproic acid (positive group). Also, oxytocin showed an increase in the frequency and time of contact between the two larvae. We showed a decrease in the distance in the larval group and an increase in time spent at a distance of one centimeter from the mirror. 
Conclusion: Our findings showed that the increased gene expression of shank3a, shank3b, and oxytocin receptors improved autistic behavior. Based on this study some indications showed that oxytocin administration in the larval stage could significantly improve the autism-like spectrum.


1. Schneider T, Przewłocki R. Behavioral alterations in rats prenatally exposed to valproic acid: animal model of autism. Neuropsychopharmacology 2005; 30:80-89.
2. Banerjee A, García-Oscos F, Roychowdhury S, Galindo LC, Hall S, Kilgard MP, et al. Impairment of cortical GABAergic synaptic transmission in an environmental rat model of autism. Int J Neuropsychopharmacol 2013; 16:1309-1318.
3. Sheldrick RC, Carter AS. State-level trends in the prevalence of Autism Spectrum Disorder (ASD) from 2000 to 2012: A reanalysis of findings from the autism and developmental disabilities network. J Autism Dev Disord 2018; 48:3086-3092.
4. Constantino JN, Todd RD. Autistic traits in the general population: a twin study. Arch Gen Psychiatry 2003; 60:524-530.
5. Weintraub K. Autism counts. Nature 2011; 479:22.
6. Zafeiriou DI, Ververi A, Dafoulis V, Kalyva E, Vargiami E. Autism spectrum disorders: The quest for genetic syndromes. Am J Med Genet B Neuropsychiatr Genet 2013; 162:327-366.
7. Constantino J, Todorov A, Hilton C, Law P, Zhang Y, Molloy E, et al. Autism recurrence in half siblings: strong support for genetic mechanisms of transmission in ASD. Mol Psychiatry 2013; 18:137-138.
8. Evans B. How autism became autism: The radical transformation of a central concept of child development in Britain. Hist Human Sci 2013; 26:3-31.
9. Mayes SD, Calhoun SL, Murray MJ, Ahuja M, Smith LA. Anxiety, depression, and irritability in children with autism relative to other neuropsychiatric disorders and typical development. Res Autism Spectr Disord 2011; 5:474-485.
10. Geschwind DH. Genetics of autism spectrum disorders. Trends Cogn Sci 2011; 15:409-416.
11. Voineagu I, Wang X, Johnston P, Lowe JK, Tian Y, Horvath S, et al. Transcriptomic analysis of autistic brain reveals convergent molecular pathology. Nature 2011; 474:380-384.
12. Muhle R, Trentacoste SV, Rapin I. The genetics of autism. Pediatrics 2004; 113:e472-e486.
13. Meshalkina DA, Kizlyk MN, Kysil EV, Collier AD, Echevarria DJ, Abreu MS, et al. Zebrafish models of autism spectrum disorder. Exp Neurol 2018; 299:207-216.
14. Benvenuto A, Battan B, Porfirio MC, Curatolo P. Pharmacotherapy of autism spectrum disorders. Brain Dev 2013; 35:119-127.
15. Farmer C, Thurm A, Grant P. Pharmacotherapy for the core symptoms in autistic disorder: current status of the research. Drugs 2013; 73:303-314.
16. Sepand MR, Ghavami M, Zanganeh S, Stacks S, Ghasemi F, Montazeri H, et al. Impact of plasma concentration of transferrin on targeting capacity of nanoparticles. Nanoscale 2020; 12:4935-4944.
17. Yamasue H, Yee JR, Hurlemann R, Rilling JK, Chen FS, Meyer-Lindenberg A, et al. Integrative approaches utilizing oxytocin to enhance prosocial behavior: from animal and human social behavior to autistic social dysfunction. J Neurosci 2012; 32:14109-14117a.
18. Van IJzendoorn MH, Bakermans-Kranenburg MJ. A sniff of trust: meta-analysis of the effects of intranasal oxytocin administration on face recognition, trust to in-group, and trust to out-group. Psychoneuroendocrinology 2012; 37:438-443.
19. Bakermans-Kranenburg M, Van Ijzendoorn M. Sniffing around oxytocin: Review and meta-analyses of trials in healthy and clinical groups with implications for pharmacotherapy. Transl Psychiatry 2013; 3:e258-e258.
20. Tachibana M, Kagitani-Shimono K, Mohri I, Yamamoto T, Sanefuji W, Nakamura A, et al. Long-term administration of intranasal oxytocin is a safe and promising therapy for early adolescent boys with autism spectrum disorders. J Child Adolesc Psychopharmacol 2013; 23:123-127.
21. Veening JG, Olivier B. Intranasal administration of oxytocin: behavioral and clinical effects, a review. Neurosci Biobehav Rev 2013; 37:1445-1465.
22. Yamasue H. Promising evidence and remaining issues regarding the clinical application of oxytocin in autism spectrum disorders. Psychiatry Clin Neurosci 2016; 70:89-99.
23. Yatawara C, Einfeld S, Hickie I, Davenport T, Guastella A. The effect of oxytocin nasal spray on social interaction deficits observed in young children with autism: a randomized clinical crossover trial. Mol Psychiatry 2016; 21:1225-1231.
24. Guastella AJ, Hickie IB. Oxytocin treatment, circuitry, and autism: a critical review of the literature placing oxytocin into the autism context. Biol Psychiatry 2016; 79:234-242.
25. Domes G, Heinrichs M, Kumbier E, Grossmann A, Hauenstein K, Herpertz SC. Effects of intranasal oxytocin on the neural basis of face processing in autism spectrum disorder. Biol Psychiatry 2013; 74:164-171.
26. Domes G, Kumbier E, Heinrichs M, Herpertz SC. Oxytocin promotes facial emotion recognition and amygdala reactivity in adults with asperger syndrome. Neuropsychopharmacology 2014; 39:698-706.
27. Carter CS, Grippo AJ, Pournajafi-Nazarloo H, Ruscio MG, Porges SW. Oxytocin, vasopressin and sociality. Prog Brain Res 2008; 170:331-336.
28. Meyer-Lindenberg A, Domes G, Kirsch P, Heinrichs M. Oxytocin and vasopressin in the human brain: social neuropeptides for translational medicine. Nat Rev Neurosci 2011; 12:524-538.
29. Sanadgol N, Barati M, Houshmand F, Hassani S, Clarner T, Golab F. Metformin accelerates myelin recovery and ameliorates behavioral deficits in the animal model of multiple sclerosis via adjustment of AMPK/Nrf2/mTOR signaling and maintenance of endogenous oligodendrogenesis during brain self-repairing period. Pharmacol Rep 2020; 72:641-658.
30. Benner S, Yamasue H. Clinical potential of oxytocin in autism spectrum disorder: current issues and future perspectives. Behav Pharmacol 2018; 29:1-12.
31. Kalueff A, Wheaton M, Murphy D. What’s wrong with my mouse model?: Advances and strategies in animal modeling of anxiety and depression. Behav Brain Res 2007; 179:1-18.
32. Kas MJ, Glennon JC, Buitelaar J, Ey E, Biemans B, Crawley J, et al. Assessing behavioural and cognitive domains of autism spectrum disorders in rodents: current status and future perspectives. Psychopharmacology 2014; 231:1125-1146.
33. Silverman JL, Oliver C, Karras M, Gastrell P, Crawley J. AMPAKINE enhancement of social interaction in the BTBR mouse model of autism. Neuropharmacology 2013; 64:268-282.
34. Silverman JL, Smith DG, Rizzo SJS, Karras MN, Turner SM, Tolu SS, et al. Negative allosteric modulation of the mGluR5 receptor reduces repetitive behaviors and rescues social deficits in mouse models of autism. Sci Transl Med 2012; 4:131ra151-131ra151.
35. Gerlai R. A small fish with a big future: zebrafish in behavioral neuroscience. Rev Neurosci 2011; 22: 3-4.
36. Kalueff AV, Stewart AM, Gerlai R. Zebrafish as an emerging model for studying complex brain disorders. Trends Pharmacol Sci 2014; 35:63-75.
37. Galeshkalami NS, Abdollahi M, Najafi R, Baeeri M, Jamshidzade A, Falak R, et al. Alpha-lipoic acid and coenzyme Q10 combination ameliorates experimental diabetic neuropathy by modulating oxidative stress and apoptosis. Life Sci 2019; 216:101-110.
38. Stewart AM, Braubach O, Spitsbergen J, Gerlai R, Kalueff AV. Zebrafish models for translational neuroscience research: from tank to bedside. Trends Neurosci 2014; 37:264-278.
39. Caramillo EM, Echevarria DJ. Alzheimer’s disease in the zebrafish: where can we take it? Behav Pharmacol 2017; 28:179-186.
40. Hill AJ, Teraoka H, Heideman W, Peterson RE. Zebrafish as a model vertebrate for investigating chemical toxicity. Toxicol Sci 2005; 86:6-19.
41. Robea MA, Ciobica A, Curpan A-S, Plavan G, Strungaru S, Lefter R, et al. Preliminary results regarding sleep in a zebrafish model of autism spectrum disorder. Brain Sci 2021; 11:556.
42. Tropepe V, Sive HL. Can zebrafish be used as a model to study the neurodevelopmental causes of autism? Genes Brain Behav 2003; 2:268-281.
43. Stewart AM, Nguyen M, Wong K, Poudel MK, Kalueff AV. Developing zebrafish models of autism spectrum disorder (ASD). Prog Neuropsychopharmacol Biol Psychiatry 2014; 50:27-36.
44. Lee S, Chun H-S, Lee J, Park H-J, Kim K-T, Kim C-H, et al. Plausibility of the zebrafish embryos/larvae as an alternative animal model for autism: A comparison study of transcriptome changes. PloS one 2018; 13:e0203543.
45. Zimmermann FF, Gaspary KV, Leite CE, Cognato GDP, Bonan CD. Embryological exposure to valproic acid induces social interaction deficits in zebrafish (Danio rerio): A developmental behavior analysis. Neurotoxicol Teratol 2015; 52:36-41.
46. Arndt TL, Stodgell CJ, Rodier PM. The teratology of autism. Int J Dev Neurosci 2005; 23:189-199.
47. Markram H, Rinaldi T, Markram K. The intense world syndrome-an alternative hypothesis for autism. Front Neurosci 2007; 1:6.
48. Liu TX, Howlett NG, Deng M, Langenau DM, Hsu K, Rhodes J, et al. Knockdown of zebrafish Fancd2 causes developmental abnormalities via p53-dependent apoptosis. Dev Cell 2003; 5:903-914.
49. Westerfield M. The zebrafish book: a guide for the laboratory use of zebrafish. http://zfin org/zf_info/zfbook/zfbk html 2000.
50. Nunes MEM, Schimith LE, da Costa-Silva DG, Lopes AR, Leandro LP, Martins IK, et al. Acute exposure to permethrin modulates behavioral functions, redox, and bioenergetics parameters and induces DNA damage and cell death in larval zebrafish. Oxid Med Cell Longev 2019; 2019: 9149203.
51. Kaushal G, Sayre BE, Prettyman T. Stability-indicating HPLC method for the determination of the stability of oxytocin parenteral solutions prepared in polyolefin bags. Drug Discov Ther 2012; 6:49-54.
52. Fleming A, Diekmann H, Goldsmith P. Functional characterisation of the maturation of the blood-brain barrier in larval zebrafish. PloS One 2013; 8:e77548.
53. Chen J, Lei L, Tian L, Hou F, Roper C, Ge X, et al. Developmental and behavioral alterations in zebrafish embryonically exposed to valproic acid (VPA): An aquatic model for autism. Neurotoxicol Teratol 2018; 66:8-16.
54. Karvat G, Kimchi T. Acetylcholine elevation relieves cognitive rigidity and social deficiency in a mouse model of autism. Neuropsychopharmacology 2014; 39:831-840.
55. Abrahams BS, Arking DE, Campbell DB, Mefford HC, Morrow EM, Weiss LA, et al. SFARI Gene 2.0: A community-driven knowledgebase for the autism spectrum disorders (ASDs). Mol Autism 2013; 4:1-3.
56. Steenbergen PJ, Richardson MK, Champagne DL. The use of the zebrafish model in stress research. Prog Neuropsychopharmacol Biol Psychiatry 2011; 35:1432-1451.
57. Taghizadeh G, Pourahmad J, Mehdizadeh H, Foroumadi A, Torkaman-Boutorabi A, Hassani S, et al. Protective effects of physical exercise on MDMA-induced cognitive and mitochondrial impairment. Free Radic Biol Med 2016; 99:11-19.
58. Maximino C, De Brito TM, de Mattos Dias CAG, Gouveia A, Morato S. Scototaxis as anxiety-like behavior in fish. Nat Protoc 2010; 5:209-216.
59. Spencer C, Alekseyenko O, Serysheva E, Yuva‐Paylor L, Paylor R. Altered anxiety‐related and social behaviors in the Fmr1 knockout mouse model of fragile X syndrome. Genes Brain Behav 2005; 4:420-430.
60. Pham M, Raymond J, Hester J, Kyzar E, Gaikwad S, Bruce I, et al. Assessing social behavior phenotypes in adult zebrafish: Shoaling, social preference, and mirror biting tests.  Zebrafish protocols for neurobehavioral research: Springer; 2012. p. 231-246.
61. Ribeiro D, Nunes AR, Gliksberg M, Anbalagan S, Levkowitz G, Oliveira RF. Oxytocin receptor signalling modulates novelty recognition but not social preference in zebrafish. J Neuroendocrinol 2020; 32:e12834.
62. Cherepanov SM, Gerasimenko M, Yuhi T, Furuhara K, Tsuji C, Yokoyama S, et al. Oxytocin ameliorates impaired social behavior in a Chd8 haploinsufficiency mouse model of autism. BMC Neurosci 2021; 22:1-13.
63. Liu Z-H, Li Y-W, Hu W, Chen Q-L, Shen Y-J. Mechanisms involved in tributyltin-enhanced aggressive behaviors and fear responses in male zebrafish. Aquat Toxicol 2020; 220:105408.
64. Beery AK. Antisocial oxytocin: complex effects on social behavior. Curr Opin Behav Sci 2015; 6:174-182.
65. Dölen G. Oxytocin: parallel processing in the social brain? J Neuroendocrinol 2015; 27:516-535.
66. Kingsbury MA, Bilbo SD. The inflammatory event of birth: How oxytocin signaling may guide the development of the brain and gastrointestinal system. Front Neuroendocrinol 2019; 55:100794.
67. Grinevich V, Neumann ID. Brain oxytocin: how puzzle stones from animal studies translate into psychiatry. Mol Psychiatry 2021; 26:265-279.
68. Dara T, Vatanara A, Sharifzadeh M, Khani S, Vakilinezhad MA, Vakhshiteh F, et al. Improvement of memory deficits in the rat model of Alzheimer’s disease by erythropoietin-loaded solid lipid nanoparticles. Neurobiol Learn Mem 2019; 166:107082.
69. Romano A, Tempesta B, Micioni Di Bonaventura MV, Gaetani S. From autism to eating disorders and more: the role of oxytocin in neuropsychiatric disorders. Front Neurosci 2016; 9:497.
70. Gemmer A, Mirkes K, Anneser L, Eilers T, Kibat C, Mathuru A, et al. Oxytocin receptors influence the development and maintenance of social behavior in zebrafish (Danio rerio). Sci Rep 2022; 12:1-13.
71. Mooney SJ, Douglas NR, Holmes MM. Peripheral administration of oxytocin increases social affiliation in the naked mole-rat (Heterocephalus glaber). Horm Behav 2014; 65:380-385.
72. Goodson JL, Schrock SE, Klatt JD, Kabelik D, Kingsbury MA. Mesotocin and nonapeptide receptors promote estrildid flocking behavior. Science 2009; 325:862-866.
73. Nishizato M, Fujisawa TX, Kosaka H, Tomoda A. Developmental changes in social attention and oxytocin levels in infants and children. Sci Rep 2017; 7:1-10.
74. Alvares GA, Quintana DS, Whitehouse AJ. Beyond the hype and hope: Critical considerations for intranasal oxytocin research in autism spectrum disorder. Autism Res 2017; 10:25-41.
75. Momtaz S, Salek-Maghsoudi A, Abdolghaffari AH, Jasemi E, Rezazadeh S, Hassani S, et al. Polyphenols targeting diabetes via the AMP-activated protein kinase pathway; future approach to drug discovery. Crit Rev Clin Lab Sci 2019; 56:472-492.
76. Ogi A, Licitra R, Naef V, Marchese M, Fronte B, Gazzano A, et al. Social preference tests in zebrafish: A systematic review. Front Vet Sci 2021; 7:1239.
77. Mogi K, Ooyama R, Nagasawa M, Kikusui T. Effects of neonatal oxytocin manipulation on development of social behaviors in mice. Physiol Behav 2014; 133:68-75.
78. Braida D, Donzelli A, Martucci R, Capurro V, Busnelli M, Chini B, et al. Neurohypophyseal hormones manipulation modulate social and anxiety-related behavior in zebrafish. Psychopharmacology 2012; 220:319-330.
79. Hovey D. On oxytocin and social behavior.  2018.
80. Pfaender S, Sauer AK, Hagmeyer S, Mangus K, Linta L, Liebau S, et al. Zinc deficiency and low enterocyte zinc transporter expression in human patients with autism related mutations in SHANK3. Sci Rep 2017; 7:1-15.
81. Landin J, Hovey D, Xu B, Lagman D, Zettergren A, Larhammar D, et al. Oxytocin receptors regulate social preference in zebrafish. Sci Rep 2020; 10:1-12.
82. Zimmermann FF, Gaspary KV, Siebel AM, Leite CE, Kist LW, Bogo MR, et al. Analysis of extracellular nucleotide metabolism in adult zebrafish after embryological exposure to valproic acid. Mol Neurobiol 2017; 54:3542-3553.
83. Liu C-x, Li C-y, Hu C-c, Wang Y, Lin J, Jiang Y-h, et al. CRISPR/Cas9-induced shank3b mutant zebrafish display autism-like behaviors. Mol Autism 2018; 9:1-13.
84. Rea V, Van Raay TJ. Using zebrafish to model autism spectrum disorder: A comparison of ASD risk genes between zebrafish and their mammalian counterparts. Front Mol Neurosci 2020; 13:575575.
85. Lee DK, Li SW, Bounni F, Friedman G, Jamali M, Strahs L, et al. Reduced sociability and social agency encoding in adult Shank3-mutant mice are restored through gene re-expression in real time. Nat Neurosci 2021; 24:1243-1255.
86. Kozol RA, Cukier HN, Zou B, Mayo V, De Rubeis S, Cai G, et al. Two knockdown models of the autism genes SYNGAP1 and SHANK3 in zebrafish produce similar behavioral phenotypes associated with embryonic disruptions of brain morphogenesis. Hum Mol Genet 2015; 24:4006-4023.
87. Song T-J, Lan X-Y, Wei M-P, Zhai F-J, Boeckers TM, Wang J-N, et al. Altered behaviors and impaired synaptic function in a novel rat model with a complete Shank3 deletion. Front Cell Neurosci 2019; 13:111.
88. Gouder L, Vitrac A, Goubran-Botros H, Danckaert A, Tinevez J-Y, André-Leroux G, et al. Altered spinogenesis in iPSC-derived cortical neurons from patients with autism carrying de novo SHANK3 mutations. Sci Rep 2019; 9:1-11.
89. Harony-Nicolas H, Kay M, du Hoffmann J, Klein ME, Bozdagi-Gunal O, Riad M, et al. Oxytocin improves behavioral and electrophysiological deficits in a novel Shank3-deficient rat. Elife 2017; 6:e18904.
90. LoParo D, Waldman I. The oxytocin receptor gene (OXTR) is associated with autism spectrum disorder: A meta-analysis. Mol Psychiatry 2015; 20:640-646.