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6-OHDA-Lesioned Adult Zebrafish as a Useful Parkinson’s Disease Model for Dopaminergic Neuroregeneration

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Abstract

Conventional mammalian models of neurodegeneration are often limited by futile axonogenesis with minimal functional recuperation of severed neurons. The emergence of zebrafish, a non-mammalian model with excellent neuroregenerative properties, may address these limitations. This study aimed to establish an adult zebrafish-based, neurotoxin-induced Parkinson’s disease (PD) model and subsequently validate the regenerative capability of dopaminergic neurons (DpN). The DpN of adult male zebrafish (Danio rerio) were lesioned by microinjecting 6-hydroxydopamine (6-OHDA) neurotoxin (6.25, 12.5, 18.75, 25, 37.5, 50 and 100 mg/kg) into the ventral diencephalon (Dn). This was facilitated by an optimised protocol that utilised 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanineperchlorate (DiI) dye to precisely identify the injection site. Immunostaining was utilised to identify the number of tyrosine hydroxylase immunoreactive (TH-ir) DpN in brain regions of interest (i.e. olfactory bulb, telencephalon, preoptic area, posterior tuberculum and hypothalamus). Open tank video recordings were performed for locomotor studies. The Dn was accessed by setting the injection angle of the microinjection capillary to 60° and injection depth to 1200 μm (from the exposed brain surface). 6-OHDA (25 mg/kg) successfully ablated >85% of the Dn DpN (preoptic area, posterior tuberculum and hypothalamus) whilst maintaining a 100% survival. Locomotor analysis of 5-min recordings revealed that 6-OHDA-lesioned adult zebrafish were significantly (p < 0.0001) reduced in speed (cm/s) and distance travelled (cm). Lesioned zebrafish showed full recovery of Dn DpN 30 days post-lesion. This study had successfully developed a stable 6-OHDA-induced PD zebrafish model using a straightforward and reproducible approach. Thus, this developed teleost model poses exceptional potentials to study DpN regeneration.

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Abbreviations

DiI:

1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanineperchlorate

MPTP:

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

DAPI:

4′,6-diamidino-2-phenylindole

6-OHDA:

6-Hydroxydopamine

ANOVA:

Analysis of variance

CARE:

Committee on Animal Research and Ethics

Dn:

Diencephalon

DpN:

Dopaminergic neurons

Hab:

Habenula

hPc:

Capillary pressure

hPi:

Injection pressure

Hyp:

Hypothalamus

ICV:

Intracerebroventricular

MPTP:

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

MO:

Medulla oblongata

OB:

Olfactory bulb

OCT:

Optimal cutting temperature

PFA:

Paraformaldehyde

PD:

Parkinson’s disease

PNS:

Peripheral nervous system

PBS:

Phosphate-buffered saline

PT:

Posterior tuberculum

POA:

Preoptic area

SN:

Substantia nigra

SNc:

Substantia nigra pars compacta

Tec:

Tectum

Tel:

Telencephalon

MS-222:

Tricaine methanesulfonate

TH:

Tyrosine hydroxylase

TH-ir:

Tyrosine hydroxylase immunoreactive

References

  • Abeliovich A, Hammond R (2007) Midbrain dopamine neuron differentiation: factors and fates. Dev Biol 304:447–454

    Article  CAS  PubMed  Google Scholar 

  • Alföldi J, Di Palma F, Grabherr M, Williams C, Kong L, Mauceli E, Russell P, Lowe CB, Glor RE, Jaffe JD (2011) The genome of the green anole lizard and a comparative analysis with birds and mammals. Nature 477:587–591

    Article  PubMed  PubMed Central  Google Scholar 

  • Anichtchik OV, Kaslin J, Peitsaro N, Scheinin M, Panula P (2004) Neurochemical and behavioural changes in zebrafish (Danio rerio) after systemic administration of 6-hydroxydopamine and 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine. J Neurochem 88:443–453

    Article  CAS  PubMed  Google Scholar 

  • Arvidsson A, Collin T, Kirik D, Kokaia Z, Lindvall O (2002) Neuronal replacement from endogenous precursors in the adult brain after stroke. Nat Med 8:963–970

    Article  CAS  PubMed  Google Scholar 

  • Avdesh A, Chen M, Martin-Iverson M, Mondal A, Ong D, Rainey-Smith S, Taddei K, Lardelli M, Groth D, Verdile G (2011) Regular care and maintenance of a zebrafish (Danio rerio) laboratory: an introduction. J Vis Exp:e4196–e4196

  • Bai Q, Mullett SJ, Garver JA, Hinkle DA, Burton EA (2006) Zebrafish DJ-1 is evolutionarily conserved and expressed in dopaminergic neurons. Brain Res 1113:33–44

    Article  CAS  PubMed  Google Scholar 

  • Barberi T, Klivenyi P, Calingasan NY, Lee H, Kawamata H, Loonam K, Perrier AL, Bruses J, Rubio ME, Topf N (2003) Neural subtype specification of fertilization and nuclear transfer embryonic stem cells and application in parkinsonian mice. Nat Biotechnol 21:1200–1207

    Article  CAS  PubMed  Google Scholar 

  • Barbosa A, Maximino C, de Souza Fim Pereira A, Wolkers CPB, Alves FL, Ide LM, Herculano AM, Hoffmann A (2012) Rapid method for acute intracerebroventricular injection in adult zebrafish. Zebrafish Protoc Neurobehav Res:323–330

  • Becker T, Becker CG (2014) Axonal regeneration in zebrafish. Curr Opin Neurobiol 27:186–191

    Article  CAS  PubMed  Google Scholar 

  • Berg DA, Kirkham M, Beljajeva A, Knapp D, Habermann B, Ryge J, Tanaka EM, Simon A (2010) Efficient regeneration by activation of neurogenesis in homeostatically quiescent regions of the adult vertebrate brain. Development 137:4127–4134

    Article  CAS  PubMed  Google Scholar 

  • Berg DA, Kirkham M, Wang H, Frisén J, Simon A (2011) Dopamine controls neurogenesis in the adult salamander midbrain in homeostasis and during regeneration of dopamine neurons. Cell Stem Cell 8:426–433

    Article  CAS  PubMed  Google Scholar 

  • Betarbet R, Sherer TB, Greenamyre JT (2002) Animal models of Parkinson’s disease. BioEssays 24:308–318

    Article  CAS  PubMed  Google Scholar 

  • Blum D, Torch S, Lambeng N, Nissou M-F, Benabid A-L, Sadoul R, Verna J-M (2001) Molecular pathways involved in the neurotoxicity of 6-OHDA, dopamine and MPTP: contribution to the apoptotic theory in Parkinson’s disease. Prog Neurobiol 65:135–172

    Article  CAS  PubMed  Google Scholar 

  • Bonito-Oliva A, Masini D, Fisone G (2014) A mouse model of non-motor symptoms in Parkinson’s disease: focus on pharmacological interventions targeting affective dysfunctions. Front Behav Neurosci 8:290

    Article  PubMed  PubMed Central  Google Scholar 

  • Botella JA, Bayersdorfer F, Gmeiner F, Schneuwly S (2009) Modelling Parkinson’s disease in Drosophila. NeuroMolecular Med 11:268–280

    Article  CAS  PubMed  Google Scholar 

  • Bove J, Perier C (2012) Neurotoxin-based models of Parkinson’s disease. Neuroscience 211:51–76

    Article  CAS  PubMed  Google Scholar 

  • Bretaud S, Lee S, Guo S (2004) Sensitivity of zebrafish to environmental toxins implicated in Parkinson’s disease. Neurotoxicol Teratol 26:857–864

    Article  CAS  PubMed  Google Scholar 

  • Collymore C, Tolwani RJ, Rasmussen S (2015) The behavioral effects of single housing and environmental enrichment on adult zebrafish (Danio rerio). J Am Assoc Lab Anim Sci 54:280–285

    PubMed  PubMed Central  Google Scholar 

  • Damier P, Hirsch E, Agid Y, Graybiel A (1999) The substantia nigra of the human brain. Brain 122:1421–1436

    Article  PubMed  Google Scholar 

  • Detrich HW III, Westerfield M, Zon L (2011) The zebrafish: cellular and developmental biology. Part B: Cell Dev Biol: Acad Press

  • Deumens R, Blokland A, Prickaerts J (2002) Modeling Parkinson’s disease in rats: an evaluation of 6-OHDA lesions of the nigrostriatal pathway. Exp Neurol 175:303–317

    Article  CAS  PubMed  Google Scholar 

  • Dias TB, Yang Y-J, Ogai K, Becker T, Becker CG (2012) Notch signaling controls generation of motor neurons in the lesioned spinal cord of adult zebrafish. J Neurosci 32:3245–3252

    Article  CAS  PubMed  Google Scholar 

  • Du Y, Guo Q, Shan M, Wu Y, Huang S, Zhao H, Hong H, Yang M, Yang X, Ren L (2016) Spatial and temporal distribution of dopaminergic neurons during development in zebrafish. Front Neuroanat 10

  • Eslamboli A, Georgievska B, Ridley RM, Baker HF, Muzyczka N, Burger C, Mandel RJ, Annett L, Kirik D (2005) Continuous low-level glial cell line-derived neurotrophic factor delivery using recombinant adeno-associated viral vectors provides neuroprotection and induces behavioral recovery in a primate model of Parkinson's disease. J Neurosci 25:769–777

    Article  CAS  PubMed  Google Scholar 

  • Flinn L, Bretaud S, Lo C, Ingham PW, Bandmann O (2008) Zebrafish as a new animal model for movement disorders. J Neurochem 106:1991–1997

    Article  CAS  PubMed  Google Scholar 

  • Geraerts M, Krylyshkina O, Debyser Z, Baekelandt V (2007) Concise review: therapeutic strategies for Parkinson disease based on the modulation of adult neurogenesis. Stem Cell 25:263–270

    Article  CAS  Google Scholar 

  • Ghosh S, Hui SP (2016) Regeneration of zebrafish CNS: adult neurogenesis. Neural Plast. doi:10.1155/2016/5815439

  • Gibrat C, Saint-Pierre M, Bousquet M, Lévesque D, Rouillard C, Cicchetti F (2009) Differences between subacute and chronic MPTP mice models: investigation of dopaminergic neuronal degeneration and α-synuclein inclusions. J Neurochem 109:1469–1482

    Article  CAS  PubMed  Google Scholar 

  • Goldshmit Y, Sztal TE, Jusuf PR, Hall TE, Nguyen-Chi M, Currie PD (2012) Fgf-dependent glial cell bridges facilitate spinal cord regeneration in zebrafish. J Neurosci 32:7477–7492

    Article  CAS  PubMed  Google Scholar 

  • Haehner A, Hummel T, Reichmann H (2011) Olfactory loss in Parkinson’s disease. Parkinsons Dis 2011. doi:10.4061/2011/450939

  • Harrington AJ, Hamamichi S, Caldwell GA, Caldwell KA (2010) C. elegans as a model organism to investigate molecular pathways involved with Parkinson’s disease. Dev Dynam 239:1282–1295

    Article  CAS  Google Scholar 

  • Herculano-Houzel S (2012) The isotropic fractionator: a fast, reliable method to determine numbers of cells in the brain or other tissues. Concepts and Experimental Approaches, Neuronal Network Analysis, pp 391–403

    Google Scholar 

  • Herculano-Houzel S, von Bartheld CS, Miller DJ, Kaas JH (2015) How to count cells: the advantages and disadvantages of the isotropic fractionator compared with stereology. Cell Tissue Res 360:29–42

    Article  PubMed  PubMed Central  Google Scholar 

  • Höglinger GU, Alvarez-Fischer D, Arias-Carrión O, Djufri M, Windolph A, Keber U, Borta A, Ries V, Schwarting RK, Scheller D (2015) A new dopaminergic nigro-olfactory projection. Acta Neuropathol 130:333–348

    Article  PubMed  Google Scholar 

  • Höglinger GU, Respondek G, Stamelou M, Kurz C, Josephs KA, Lang AE, Mollenhauer B, Müller U, Nilsson C, Whitwell JL (2017) Clinical diagnosis of progressive supranuclear palsy: the movement disorder society criteria. Movement Disord

    Google Scholar 

  • Howe K, Clark MD, Torroja CF, Torrance J, Berthelot C, Muffato M, Collins JE, Humphray S, McLaren K, Matthews L (2013) The zebrafish reference genome sequence and its relationship to the human genome. Nature 496:498–503

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jackson-Lewis V, Blesa J, Przedborski S (2012) Animal models of Parkinson’s disease. Parkinsonism Relat D 18:S183–S185

    Article  Google Scholar 

  • Kalueff AV, Gebhardt M, Stewart AM, Cachat JM, Brimmer M, Chawla JS, Craddock C, Kyzar EJ, Roth A, Landsman S (2013) Towards a comprehensive catalog of zebrafish behavior 1.0 and beyond. Zebrafish 10:70–86

    Article  PubMed  PubMed Central  Google Scholar 

  • Kirkham M, Joven A (2015) Studying newt brain regeneration following subtype specific neuronal ablation. Salamander Regen Res: Method Protoc 1290:91–99

    Google Scholar 

  • Kizil C, Iltzsche A, Kaslin J, Brand M (2013) Micromanipulation of gene expression in the adult zebrafish brain using cerebroventricular microinjection of morpholino oligonucleotides. J Vis Exp:e50415–e50415

  • Kostrzewa RM, Jacobowitz DM (1974) Pharmacological actions of 6-hydroxydopamine. Pharmacol Rev 26:199–288

    CAS  PubMed  Google Scholar 

  • Lee SW, Hu YS, Hu LF, Lu Q, Dawe GS, Moore PK, Wong PTH, Bian JS (2006) Hydrogen sulphide regulates calcium homeostasis in microglial cells. Glia 54:116–124

    Article  PubMed  Google Scholar 

  • Liu J-C, Koppula S, Huh S-J, Park P-J, Kim C-G, Lee C-J, Kim C-G (2015) Necrosis inhibitor-5 (NecroX-5), attenuates MPTP-induced motor deficits in a zebrafish model of Parkinson’s disease. Genes Genomics 37:1073–1079

    Article  CAS  Google Scholar 

  • Ma PM (2003) Catecholaminergic systems in the zebrafish. IV Organization and projection pattern of dopaminergic neurons in the diencephalon. J Comp Neurol 460:13–37

    Article  CAS  PubMed  Google Scholar 

  • Maetzler W, Liepelt I, Berg D (2009) Progression of Parkinson’s disease in the clinical phase: potential markers. Lancet Neurol 8:1158–1171

    Article  CAS  PubMed  Google Scholar 

  • McKinley ET, Baranowski TC, Blavo DO, Cato C, Doan TN, Rubinstein AL (2005) Neuroprotection of MPTP-induced toxicity in zebrafish dopaminergic neurons. Mol Brain Res 141:128–137

    Article  CAS  PubMed  Google Scholar 

  • Müller B, Assmus J, Herlofson K, Larsen JP, Tysnes O-B (2013) Importance of motor vs. non-motor symptoms for health-related quality of life in early Parkinson’s disease. Parkinsonism Relat D 19:1027–1032

    Article  Google Scholar 

  • Parish CL, Beljajeva A, Arenas E, Simon A (2007) Midbrain dopaminergic neurogenesis and behavioural recovery in a salamander lesion-induced regeneration model. Development 134:2881–2887

    Article  CAS  PubMed  Google Scholar 

  • Parker MO, Brock AJ, Walton RT, Brennan CH (2014) The role of zebrafish (Danio rerio) in dissecting the genetics and neural circuits of executive function. Front Neural Circuits 7:63–63

    Google Scholar 

  • Pienaar IS, Götz J, Feany MB (2010) Parkinson’s disease: insights from non-traditional model organisms. Prog Neurobiol 92:558–571

    Article  CAS  PubMed  Google Scholar 

  • Pont-Sunyer C, Hotter A, Gaig C, Seppi K, Compta Y, Katzenschlager R, Mas N, Hofeneder D, Brücke T, Bayés A (2015) The onset of nonmotor symptoms in Parkinson’s disease (the ONSET PD study). Movement Disord 30:229–237

    Article  PubMed  Google Scholar 

  • Potashkin J, Blume S, Runkle N (2010) Limitations of animal models of Parkinson’s disease. Parkinsons Dis 2011. doi:10.4061/2011/658083

  • Reimer MM, Kuscha V, Wyatt C, Sörensen I, Frank RE, Knüwer M, Becker T, Becker CG (2009) Sonic hedgehog is a polarized signal for motor neuron regeneration in adult zebrafish. J Neurosci 29:15073–15082

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Reimer MM, Sörensen I, Kuscha V, Frank RE, Liu C, Becker CG, Becker T (2008) Motor neuron regeneration in adult zebrafish. J Neurosci 28:8510–8516

    Article  CAS  PubMed  Google Scholar 

  • Rink E, Wullimann MF (2002) Connections of the ventral telencephalon and tyrosine hydroxylase distribution in the zebrafish brain (Danio rerio) lead to identification of an ascending dopaminergic system in a teleost. Brain Res Bull 57:385–387

    Article  CAS  PubMed  Google Scholar 

  • Risner ML, Lemerise E, Vukmanic EV, Moore A (2006) Behavioral spectral sensitivity of the zebrafish (Danio rerio). Vis Res 46:2625–2635

    Article  PubMed  Google Scholar 

  • Sarath Babu N, Murthy CLN, Kakara S, Sharma R, Swamy B, Cherukuvada V, Idris MM (2016) 1-Methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine induced Parkinson’s disease in zebrafish. Proteomics 16:1407–1420

    Article  CAS  PubMed  Google Scholar 

  • Schmidt R, Beil T, Strähle U, Rastegar S (2014) Stab wound injury of the zebrafish adult telencephalon: a method to investigate vertebrate brain neurogenesis and regeneration. J Vis Exp:e51753–e51753

  • Schober A (2004) Classic toxin-induced animal models of Parkinson’s disease: 6-OHDA and MPTP. Cell Tissue Res 318:215–224

    Article  PubMed  Google Scholar 

  • Schweitzer J, Driever W (2009) Development of the dopamine systems in zebrafish. In: Development and engineering of dopamine neurons, pp 1-14: Springer

  • Tan LC (2012) Mood disorders in Parkinson’s disease. Parkinsonism Relat D 18:S74–S76

    Article  Google Scholar 

  • Tenreiro S, Outeiro TF (2010) Simple is good: yeast models of neurodegeneration. FEMS Yeast Res 10:970–979

    Article  CAS  PubMed  Google Scholar 

  • Thiele SL, Warre R, Nash JE (2012) Development of a unilaterally-lesioned 6-OHDA mouse model of Parkinson’s disease. J Vis Exp 60:pii: 3234. doi:10.3791/3234

  • Tieu K (2011) A guide to neurotoxic animal models of Parkinson’s disease. Cold Spring Harbor Perspec Med 1:a009316. doi:10.1101/cshperspect.a009316

  • Valle-Leija P, Drucker-Colín R (2014) Unilateral olfactory deficit in a hemiparkinson’s disease mouse model. Neuroreport 25:948–953

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by the Ministry of Higher Education Malaysia [ 600-RMI/FRGS 5/3 (0078/2016) ].

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Author notes

  1. Yuganthini Vijayanathan and Fei Tieng Lim contributed equally to the writing of this article.

Authors and Affiliations

  1. Collaborative Drug Discovery Research (CDDR) Group, Faculty of Pharmacy, Universiti Teknologi MARA (UiTM), 42300, Puncak Alam, Selangor Darul Ehsan, Malaysia

    Yuganthini Vijayanathan, Fei Tieng Lim, Siong Meng Lim, Abu Bakar Abdul Majeed & Kalavathy Ramasamy

  2. Division of Geriatric Medicine, Department of Medicine, Faculty of Medicine, University of Malaya, 50603, Kuala Lumpur, Malaysia

    Yuganthini Vijayanathan & Maw Pin Tan

  3. School of Pharmacy, KPJ Healthcare University College, 71800, Nilai, Negeri Sembilan, Malaysia

    Chiau Ming Long

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  1. Yuganthini Vijayanathan
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  2. Fei Tieng Lim
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Correspondence to Kalavathy Ramasamy.

Ethics declarations

The present animal study was approved by the Committee on Animal Research and Ethics (CARE), Universiti Teknologi MARA (UiTM) [Reference No: 104/2015; dated 19 August 2015].

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The authors declare that they have no conflicts of interest.

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Vijayanathan, Y., Lim, F.T., Lim, S.M. et al. 6-OHDA-Lesioned Adult Zebrafish as a Useful Parkinson’s Disease Model for Dopaminergic Neuroregeneration. Neurotox Res 32, 496–508 (2017). https://doi.org/10.1007/s12640-017-9778-x

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