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Show THE UNIVERSITY OF UTAH HONORS PROGRAM Standardizing Dopaminergic Neuronal Histology in Drosophila Melanogaster B. Welch (D. Drobysheva, K. Ameel, K. Chaichana, A. Schmid) University of Utah Eccles Institute of Human Genetics Parkinson Disease (PD) is characterized by the dopaminergic neuronal loss in the brain due to environmental, genetic, and toxin-induced causes. Animals with PD exhibit common physiological symptoms such as uncontrollable tremor, inability to initiate movement, and slowness, eventually leading to death. Dopaminergic neuronal loss can be documented histologically in all animal models with PD and is a key requirement for accurate quantification of dopaminergic loss, and thus, the progression of the disease. To date, the best histological techniques for the detection of dopaminergic neurons have produced inconsistent results in Drosophila, inconsistencies that have called into question the validity of the best genetic models of Parkinson’s Disease. Here we report a systematic comparison of all commonly used histological techniques for the detection of dopaminergic neurons in invertebrates. The main areas of comparison are: choice of fixative, method of fixative infiltration, and embedding and sectioning protocols. We found that an optimal protocol incorporates a new fix known as Irina’s Fix with microwave vacuum infiltration and agarose embedding. Additional testing demonstrated that increasing the permeability with greater concentrations of Triton in rinses and improving the delivery of antibodies to non-vascularized neuronal tissues with vacuum resulted in an optimal histological method. All experiments used the same antibodies at identical concentrations for immunocytochemistry. This optimized protocol consistently produced images showing a greater number of cell bodies than had been previously reported, as well as axonal arborizations that have never been reported. We are now using this technique to evaluate animals demonstrating impressive Parkinsonian behavioral defects after dosing with a known dopaminergic toxin, the pesticide rotenone. We hope to use this model of PD in a screen for modifiers of pesticide susceptibility. B. Welch A. Schmid 132 |