The authors would like to thank Qiuli Wang, Language Resource Center, Colby College, for technical assistance with video processing and Eric Thomas, department of music, Colby College, for providing the background music. This project was supported by grants from the National Center for Research Resources, INBRE (P20RR016463-12), the National Institute for General Medical Sciences (P20 GM103423-12), Nationals Institutes of Health and Science Division Grant, Colby College (STA). JL and LWM were supported by grants from Summer Scholar Fund, Colby College.

1. Drosophila Startle-induced Locomotion Assay
<ol>
	<li>Drug Treatment
	<ol>
		<li>Sedate to immobilize desired number (approximately 8-12) of 1-3 day-old male flies using CO2 and transport them to vials containing the drug-supplemented food. Note: Another anesthetic e.g., ether or ice can be used to sedate flies to enable counting and handling.</li>
		<li>Allow flies to recover from sedation for 20 min (or until recovery) with the vial in a horizontal position (to prevent flies getting st...

<ol>
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The authors have nothing to disclose.

In this study, we describe two procedures for measuring both long-term spontaneous locomotion and short-term startle-induced locomotion in a rotenone-induced Drosophila model of Parkinson&#8217;s disease. One can also measure these locomotion characteristics in flies exposed to other pharmacological agents known to model Parkinson&#8217;s disease e.g., paraquat14, genetic models of Parkinson&#8217;s disease e.g., alpha-synuclein mutants15, and other fly models of diseases ...

Locomotion deficiencies are a major symptom of Parkinson&#8217;s disease and are largely caused by deterioration of dopaminergic neurons of the substantia nigra1. Rotenone is a ketonic insecticide that has been studied extensively to model Parkinson&#8217;s motor deficits in Drosophila2-6. Rotenone causes oxidative damage by blocking the oxidative phosphorylation pathway, which ultimately causes cell death7. Dopaminergic neurons are more prone to rotenone toxicity, making the effects of the chemical primarily motor based2,7. By inducing Parkinson&#8217;s disease symptoms in flies, we can better understand the disease and remedy its symptoms6,8-11. Drosophila provides a good model for studying this effect because they are genetically tractable, easy to maintain, and have a rapid life cycle.
Several studies have shown that rotenone causes short-term startle-induced locomotion defects in Drosophila&#8212;when flies are maintained on rotenone-supplemented food, they show a slower negative geotactic response after startle2-6. Their failure to climb upwards in a vial apparatus as quickly as control trials is indicative of startle-induced locomotion defects.
The effect of rotenone on long-term, spontaneous movement is not well described. Drosophila activity monitors (DAMs) have been successfully used to monitor movement in Drosophila circadian rhythm studies12,13. Flies are placed in individual tubes, which are loaded into the DAM. This apparatus is equipped with an infrared sensor, which counts the number of times a fly breaks the infrared beam. These counts can be used as a measure of undisturbed locomotion and activity12,13. By placing flies in a DAM, the effect of rotenone on their long-term locomotion can be characterized. This study describes methods to measure short-term startle-induced locomotion and long-term spontaneous locomotion in order to better understand the effects of rotenone mediated motor deficiencies. Characterization of locomotion deficiencies mimicking Parkinson&#8217;s disease are important because they allow for the study of other compounds which may reverse these locomotion defects.

<table border="0" cellpadding="0" cellspacing="0" width="628">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Standard narrow vials</td>
			<td style="width:127px;">Genesee Scientific</td>
			<td style="width:119px;">32-120</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Rotenone</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:119px;">R8875</td>
			<td>Store in freezer, make fresh for each experiment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Dimethyl Sulfoxide (DMSO)</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:119px;">D8418</td>
			<td>Solvent for rotenone</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Instant Drosophila medium</td>
			<td style="width:127px;">Carolina Biological&#160;</td>
			<td style="width:119px;">Formula 4-24</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Drosophila activity monitor (DAM)</td>
			<td style="width:127px;">Trikinetics</td>
			<td style="width:119px;">DAM2</td>
			<td>trikinetics.com</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">DAM tubes</td>
			<td style="width:127px;">Trikinetics</td>
			<td style="width:119px;">Tubes 5X65 mm</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;"></td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Recipe for Rotenone +food (125 mM dose)</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td>Make 62.5 mM rotenone stock solution in DMSO by dissolving 25 mg rotenone in 1 ml DMSO&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td>For 125 mM dose, add 10 mM rotenone stock in DMSO to 5 ml water.</td>
		</tr>
	</tbody>
</table>

methods to characterize spontaneous and startle-induced locomotion in a rotenone-induced parkinson's disease model of drosophila

Parkinson&#8217;s disease is a neurodegenerative disorder that results from the degeneration of dopaminergic neurons in the central nervous system, primarily in the substantia nigra. The disease causes motor deficiencies, which present as rigidity, tremors and dementia in humans. Rotenone is an insecticide that causes oxidative damage by inhibiting the function of the electron transport chain in mitochondria. It is also used to model Parkinson&#8217;s disease in the Drosophila. Flies have an inherent negative geotactic response, which compels them to climb upwards upon being startled. It has been established that rotenone causes early mortality and locomotion defects that disrupt the flies&#8217; ability to climb after they have been tapped downwards. However, the effect of rotenone on spontaneous movement is not well documented. This study outlines two sensitive, reproducible, and high throughput assays to characterize rotenone-induced deficiencies in short-term startle-induced locomotion and long-term spontaneous locomotion in Drosophila. These assays can be conveniently adapted to characterize other Drosophila models of locomotion defects and efficacy of therapeutic agents.

Drosophila Startle-induced Locomotion Assay
Wildtype, canton-S, flies showed a robust negative geotactic response with only approximately 88% and 5% of flies in the top and bottom sections respectively, of the double-vial apparatus after 30 sec (Figure 1). Flies exposed to 125 &#956;M and 250 &#956;M rotenone for 3 days showed a slight decrease in the number of flies in the top section and slight increase in the number of fli...

Watch this Scientific Journal Video about Methods to Characterize Spontaneous and Startle-induced Locomotion in a Rotenone-induced Parkinson's Disease Model of Drosophila at JoVE.com

Methods to Characterize Spontaneous and Startle-induced Locomotion in a Rotenone-induced Parkinson&#039;s Disease Model of Drosophila

Parkinson&#8217;s disease is a neurodegenerative disorder that results from the degeneration of dopaminergic neurons in the central nervous system, causing locomotion defects. Rotenone models Parkinson&#8217;s disease in Drosophila. This paper outlines two assays that characterize both spontaneous and startle-induced locomotion deficiencies caused by rotenone.

Methods to Characterize Spontaneous and Startle-induced Locomotion in a Rotenone-induced Parkinson's Disease Model of Drosophila

Parkinson&#8217;s disease is a neurodegenerative disorder that results from the degeneration of dopaminergic neurons in the central nervous system, ...

methods-to-characterize-spontaneous-startle-induced-locomotion

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JoVE Journal

Neuroscience

9.9K visualizações.  Colby College.  A doença de Parkinson é uma desordem neurodegenerativa que resulta da degeneração dos neurónios dopaminérgicos no sistema nervoso central, causando defeitos de locomoção. Modelos rotenona doença de Parkinson em Drosophila. Este documento descreve dois ensaios que caracterizam as deficiências de locomoção tanto espontâneas e induzidas por assustar causadas por rotenona. 