Function, regeneration and neuroprotection of dopaminergic neurons in the zebrafish
Davies, Nicholas Oliver
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The zebrafish has an amazing capacity for regeneration which includes regeneration of neurons within the central nervous system (CNS) both during development and into adulthood. This attribute makes the zebrafish a valuable tool in the study of regeneration. In this thesis, the research focussed on the regeneration of a specific type of cell in the CNS, dopaminergic (DA) neurons. The DA system of the zebrafish is believed to be evolutionarily conserved with comparable DA populations found in the brain of mammals. Dissimilar to mammals, however, the zebrafish is capable of regenerating various types of neurons and their axons. Thus, the zebrafish DA system provides an excellent model to study replacement of this specific and important cell type in the adult CNS. We have developed a novel toxin ablation paradigm to specifically ablate select groups of DA neurons in the adult zebrafish diencephalon, leaving other DA populations unaffected. To do this a selective DA toxin, 6-hydroxydopamine, was used. One of the ablated DA diencephalic populations is the only source of dopaminergic spinal innervation in the zebrafish. Their ablation leads to a loss of DA spinal axons following our toxin ablation. The ascending projection of the diencephalic population ablated by the toxin has been suggested as the most likely candidate for a zebrafish equivalent of the mammalian nigro-striatal pathway. The loss of cells is very specific and reproducible, indicating that these cells are particularly vulnerable to the toxin. Quantification of affected populations at various time-points post ablation was carried out to determine the capacity for regeneration of DA neurons in the CNS of zebrafish. This revealed that in some populations neuron numbers returned to those seen in controls. However, in other populations neuron numbers only partially recovered even at late time points. We have shown that this recovery is due to neurogenesis; furthermore, by inducing inflammation after the toxin treatment the recovery of DA cell numbers was accelerated by 50%. Regenerated cells originated from Olig2 positive ependymo-radial glial cells found bordering the diencephalic ventricle. We aimed to investigate the function of this group of ablated neurons through a battery of behavioural tests. These tests revealed deficits in the toxin treated animals’ fine movement, such as is necessary for maintaining shoal cohesion and breeding behaviours, whereas general movement behaviours were not found to be impaired. Zebrafish embryos also present as a great resource in the screening of drugs. Their fast and well characterised early development makes them an ideal tool for investigating previously untested neuroprotectants. A reproducible ablation paradigm similar to that established in the adults was also established in the zebrafish embryo. This was then used as a tool to investigate potential novel neuroprotectants. This screen revealed two new flavonoid compounds which had the ability to induce full protection of the affected dopaminergic cells in the zebrafish embryonic brain. The embryonic ablation model therefore represents a vertebrate in vivo model system for future high throughput screening of neuroprotective compounds against toxin induced DA cell loss. Ultimately, understanding how zebrafish functionally regenerate dopaminergic neurons using this ablation model will likely provide a useful tool into the research of neurodegenerative diseases, such as PD.