Optimizing 6-Hydroxydopamine Concentrations for induction of a Parkinson's Disease Like Behavior in Zebrafish.

Parkinson's disease is an increasing problem that affects the quality of life for over 10 million people worldwide. Affected individuals undergo worsening symptoms that begin with motor function impairment and can eventually result in death. Parkinson's Disease is associated with the loss of Tyrosine Hydroxylase (TH) expressing dopaminergic neurons. TH is the rate limiting enzyme in catecholamine neurotransmitter synthesis including dopamine. Therefore, chemically induced animal models have focused on mitigation of movement abnormalities and loss of dopaminergic neurons as indicators for drug screening. We chose the 6-hydroxydopamine (6-OHDA) chemically induced Parkinson's Disease model in zebrafish as a tool for drug screening. Previous work on this model had been conducted with varying drug concentrations and methodologies. All demonstrated reductions in TH expressing neurons; however, the behavioral assessments varied in both method, dose of OHDA (1-750 μM 6OHDA), and whether behavioral abnormalities were observed. The objective of the current study is to determine the minimal 6-OHDA dosage that is sufficient to induce a significant difference in behavioral effects while minimizing morphological toxicity. Zebrafish larvae were treated daily with 6-OHDA with ascorbic acid from 2 to 5-day post fertilization (dpf). We observed significant difference in behavior at ~20 μM 6-OHDA, and developmental delays in swim bladder formation at 85 μM 6-OHDA. The swim bladders were developed by 5 dpf when we assessed swim behavior; however, other signs of delay may still be present that would complicate results. Several studies focused on doses of 150 μM 6-OHDA or higher; however, at these doses we observed severe morphological issues including edema, curved spines, and death. Future work will focus on the TH-expressing neuron pathology as assessed by immunohistochemical and Western blotting methodologies.