The Effects of 6-hydroxydopamine (6-OHDA) and Neuronal Nitric Oxide Synthase Inhibitor ( nNOSI) on Ability of Zebrafish to recover from parkinson’s disease-like symptoms Meredith Baldasare ‘16, Joel Hoisington ‘15, and Bridgette Pouliot ’16 BI-420W-01 Seminar Research Presentation Biology Department Virginia Military Institute

Introduction Parkinson’s Disease (PD) is the 2nd most common neurodegenerative condition in the world. It is caused by the loss of dopaminergic neurons in the substantia nigra and presence of Lewy bodies in the remaining dopamine (DA) neurons. Dopamine is a catecholamine that regulates hormones, motor control and learning. Symptoms of PD include: shaking, rigidity, slowness of movement, difficulty walking, balance problems, dementia, depression, in addition to sensory, sleep, and emotional problems.

Introduction cont. Nitric oxide is a gaseous molecule that acts as an intracellular messenger for body functions such as: blood blow, platelet activity, and heart and neural/motor activity (Lorenc-Koci and Czarnecka, 2013). Neuronal nitric oxide synthase inhibitor (nNOSI) blocks nitric oxide synthesis which inhibits the dopamine pathway and causes Parkinson’s-like symptoms in zebrafish. 6-hydroxydopamine (6OHDA) is a neurotoxin that compromises dopamine neurons in the zebrafish (Parng et al., 2007). Zebrafish are a good model for our experiment because of their transparency and ability to treat them by changing their environment and have been established a a model for studying PD-like symptoms (Flinn et al., 2008).

Hypothesis If the zebrafish are treated with a co-treatment of 50μM of nNOSI and 500μM of 6OHDA, then motor deficiencies will be induced that resembles the effects of PD in humans (the listless condition). The longer the co-treatment the more difficult it will be for fish to recover from these motor deficiencies upon co-treatment cessation due to DA neuronal degeneration. Goal – to ultimately develop a zebrafish model of regenerative recovery from PD–like symptoms

Materials and Methods Zebrafish (6 days post-fertilization) were kept at 28.5°C in an incubator and their solutions that they lived in were changed out every 24 hours. Fish were counted in each well and were exposed to the treatment of either 50 µM nNOSI, 500 µM 6OHDA, or a co-treatment of both. The control fish were treated with ERS. Methylene blue was added to all solutions to keep microbes from growing. The “listless” condition was determined by whether or not the fish responded to probing with a pipet, irregular swimming patterns, if the fish demonstrated vestibular problems by sitting on it’s side, or not spontaneously swimming at all.

Materials and Methods Data analysis The percent of fish within a population that demonstrated the listless characteristics (PD-like symptoms) were determined at each 24 hour period during the assay. Data were analyzed for significant differences either by a z-test for two population proportions or for multiple proportions using a chi-square contingency table test followed by a Marascuilo's post hoc analysis

Results Figure 1 – Indicates the effects of various concentrations of nNOSI and its subsequent washout on the listless (PD-like symptoms) condition. A concentration of 50 µM elicits the maximum response over the first 24 hours of treatment. Also note that All fish recover from the listless condition after ERS washout (arrows).

Results Compare p values at 48 hours of treatment; use asterisks to indicate significant differences in survival rates. Figure 2 – Shows how the co-treatment (nNOSI + 6-OHDA) allows the fish to demonstrate PD-like symptoms with significantly greater survival than that of the single treatments (p< ---).

Results Do stats Figure 4 – Demonstrates the effect of prolonged co-treatment (nNOSI + 6OHDA) prior to ERS washout (arrows) on recovery from the listless (PD-like symptoms) condition. Note that the longer the treatment prior to washout the longer it takes the fish to recover.

Conclusions Co-treated fish survive longer than those treated in either nNOSI or 6-OHDA alone and is a better approach to establishing a regenerative recovery model. The longer the fish are exposed to the co-treatment (50uM nNOSI and 500uM of 6-OHDA) the slower their rate of recovery from PD-like symptoms after ERS washout.

Future Studies Further research could include a longer recovery period at 72 hours after ERS washout to determine if fish do slowly recover from the PD-like symptoms.

References Flinn L, Bretaud S, Lo C, Ingham PW, Bandmann O. J. 2008. Zebrafish as a new animal model for movement disorders. Neurochem. 106(5):1991-7 Lorenc-Koci E, Czarnecka A. 2013. Role of nitric oxide in the regulation of motor function. An overview of behavioral, biochemical and histological studies in animal models. Pharmacol Rep. 65(5):1043-55. Parng C, Roy NM, Ton C, Lin Y, McGrath P. 2007. Neurotoxicity assessment using zebrafish. J. Pharmacol Toxicol Methods. 55(1):103-12.

Acknowledgements COL. James Turner, Mrs. Julie Lozier, Branden Barbery, and the Virginia Military Institute Biology Department