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Characterization of Optically and Chemically Stimulated Neurotransmitter Release in Drosophila melanogaster948 views
Author
Pyakurel, Poojan, Chemistry - Graduate School of Arts and Sciences, University of Virginia
Advisors
Venton, Barbara, Department of Chemistry, University of Virginia
Abstract
Drosophila melanogaster is a popular model organism to study neurotransmission and neurological disorders. There is a wide range of genetic manipulation tools available for use in Drosophila, which has homologs for 75% of human disease genes. In addition, several fundamental neurological processes are conserved between the two species. Drosophila is therefore an excellent model organism for high throughput screening of genes involved in diseases and of drugs that can be used to treat the diseases. Since many neurological diseases are caused by changes in neurotransmitters, it is imperative to understand the fundamentals of neurochemistry and how it changes during a disease process. Electrochemical methods have long been used to measure neurotransmitters and their dynamics in the brain. Many neurotransmitters such as dopamine and octopamine are electroactive and can be measured directly on electrochemical sensors as described in this thesis.
Fast-scan cyclic voltammetry (FSCV) is a method that can provide rapid, sensitive, and selective measurements of neurotransmitters in the brain. This thesis describes new methods to stimulate endogenous dopamine release in Drosophila larval and adult CNS, and optimization of the FSCV waveform for measurements of endogenous octopamine in larvae. Chapter 2 describes acetylcholine, nicotine, and neonicotinoid stimulated dopamine release in Drosophila larval VNC. Since acetylcholine acts on endogenous Drosophila receptor nAChR to stimulate dopamine release, this method can be used to study release in most Drosophila lines. The method was also used to make the first measurements of evoked dopamine release in adult Drosophila brains in our lab. Chapter 3 describes stimulated dopamine and dopamine tissue content measurements in the CNS of adult controls and Parkinson’s disease model Drosophila with a knockdown of Parkin or RNF11 with RNAi. We found that aging does not affect the concentration of stimulated dopamine release, or the tissue content in the CNS, but has an effect when stimulations are repeated at short intervals. The release in old adults declines significantly slower than in mid-age adults. This effect was lost in Parkinson’s disease mutants suggesting that the mutations change dopamine dynamics in old adults. In Chapter 4, I describe optimization of the FSCV waveform for the detection of octopamine in situ. The new waveform uses a higher switching potential and a slower scan rate than the previously developed waveform for octopamine detection in vitro. With the new waveform, octopamine oxidation peak is detected away from the switching potential, where the background signal is most unstable. The waveform was used to measure and characterize light or ATP mediated octopamine release in Drosophila larval VNC. The methods described in this thesis enable measurements of stimulated dopamine release in most Drosophila larvae and adults, and stimulated octopamine release in larvae expressing transgenic ion channels. Future studies can investigate the effects of disease or other mutations on the release and clearance of these neurotransmitters.
Pyakurel, Poojan. Characterization of Optically and Chemically Stimulated Neurotransmitter Release in Drosophila melanogaster. University of Virginia, Chemistry - Graduate School of Arts and Sciences, PHD (Doctor of Philosophy), 2017-07-31, https://doi.org/10.18130/V3QV26.
