Name: an automated rapid iterative negative geotaxis assay for analyzing adult climbing behavior in a drosophila model of neurodegeneration
Uploaded: 2017-09-12T15:00:00
Description: Watch this Scientific Journal Video about An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration at JoVE.com

We thank the Bloomington Stock Center and the Vienna Drosophila RNAi Center for fly stocks. The patent for the RING apparatus belongs to Shanghai Advanced Research institute, Chinese Academy of Sciences. Requests for the RflyDetection software should be made to Fu-de Huang (see author list). This work was supported by grants from National Basic Research Program of China (973 Program 2013CB945602) and National Natural Science Foundation of China (31270825 and 31171043). We thank Ho lab members for discussion and comments.

1. Fly Collection
<ol>
	<li>Maintain the flies on standard fly food at 25 &#176;C, 70% humidity, and a 12 h/12 h light/dark cycle.</li>
	<li>Collect UAS-Dicer2; DDC-GAL4 fly virginsunder carbon dioxide (CO2) anesthesia.</li>
	<li>Cross these virgins to 2 day old adult male flies carrying the following genotypes: UAS-mCD8GFP (control), auxR16182 (aux RNAi), auxGFP (aux overexpression), and auxR16182; auxGFP (rescue), with a m...

<ol>
	<li>Ali, Y. O., Escala, W., Ruan, K., Zhai, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+locomotor,+learning,+and+memory+deficits+in+Drosophila+models+of+neurodegeneration.">Assaying locomotor, learning, and memory deficits in Drosophila models of neurodegeneration.</a> J Vis Exp. (49), (2011).</li><li>Nichols, C. D., Becnel, J., Pandey, U. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+assay+Drosophila+behavior.">Methods to assay Drosophila behavior.</a> J Vis Exp. (61), (2012).</li><li>Madabattula, S. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Analysis+of+Climbing+Defects+in+a+Drosophila+Model+of+Neurodegenerative+Disorders.">Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders.</a> J Vis Exp. (100), e52741 (2015).</li><li>Podratz, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+climbing+apparatus+to+measure+chemotherapy-induced+neurotoxicity+in+Drosophila+melanogaster.">An automated climbing apparatus to measure chemotherapy-induced neurotoxicity in Drosophila melanogaster.</a> Fly (Austin). 7 (3), 187-192 (2013).</li><li>Gargano, J. W., Martin, I., Bhandari, P., Grotewiel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+iterative+negative+geotaxis+(RING):+a+new+method+for+assessing+age-related+locomotor+decline+in+Drosophila.">Rapid iterative negative geotaxis (RING): a new method for assessing age-related locomotor decline in Drosophila.</a> Exp Gerontol. 40 (5), 386-395 (2005).</li><li>Liu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+rapid+iterative+negative+geotaxis+assay+and+its+use+in+a+genetic+screen+for+modifiers+of+Abeta(42)-induced+locomotor+decline+in+Drosophila.">Automated rapid iterative negative geotaxis assay and its use in a genetic screen for modifiers of Abeta(42)-induced locomotor decline in Drosophila.</a> Neurosci Bull. 31 (5), 541-549 (2015).</li><li>Shen, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SH2B1+is+Involved+in+the+Accumulation+of+Amyloid-beta42+in+Alzheimer's+Disease.">SH2B1 is Involved in the Accumulation of Amyloid-beta42 in Alzheimer's Disease.</a> J Alzheimers Dis. 55 (2), 835-847 (2017).</li><li>Song, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auxilin+Underlies+Progressive+Locomotor+Deficits+and+Dopaminergic+Neuron+Loss+in+a+Drosophila+Model+of+Parkinson's+Disease.">Auxilin Underlies Progressive Locomotor Deficits and Dopaminergic Neuron Loss in a Drosophila Model of Parkinson's Disease.</a> Cell Rep. 18 (5), 1132-1143 (2017).</li><li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Downregulation+of+RBO-PI4KIII&#945;+Facilitates+A&#946;42+Secretion+and+Ameliorates+Neural+Deficits+in+A&#946;42-Expressing+Drosophila.">Downregulation of RBO-PI4KIII&#945; Facilitates A&#946;42 Secretion and Ameliorates Neural Deficits in A&#946;42-Expressing Drosophila.</a> J Neurosci. 37 (19), 4928-4941 (2017).</li><li>Riemensperger, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+dopamine+pathway+underlies+progressive+locomotor+deficits+in+a+Drosophila+model+of+Parkinson+disease.">A single dopamine pathway underlies progressive locomotor deficits in a Drosophila model of Parkinson disease.</a> Cell Rep. 5 (4), 952-960 (2013).</li></ol>

The automated RING assay described here enables a high-throughput analysis of fly negative geotaxis behavior for hundreds of flies simultaneously. Previously existing strategies for analyzing adult climbing involve observations of a single fly in an individual vial, and fly position is manually detected by eye. This rather tedious process might sometimes cause misreading or misinterpretation of data, as well as labor-intensive work. Our automated RING assay starts with a simple click, and the apparatus automatically sync...

A variety of protocols and methods have been developed for analyzing Drosophila adult climbing behavior. Rather laborious, the traditional analysis mostly involves putting a single fly into an individual vial and uses a manual force to tap flies down for synchronization1,2,3,4. It is tedious and time consuming, unsuitable for large high-throughput studies, and has potential variations of the manual force used to tap down the flies as well as other limitations. To improve the assay, a Rapid Iterative Negative Geotaxis (RING) assay was developed which allows high-throughput analysis over numerous flies at the same time5. However, the assay still requires a manually exerting force to synchronize fly action. Our version of the RING assay, revised upon the previous assay, includes a metal base hosting multiple fly-containing vials automatically controlled by an electric motor to drive fly synchronization6. Upon recording, the fly climbing immediately after synchronization is recorded then analyzed using a self-designed software. Our automated RING assay has eliminated the tedious and labor-intensive process in collecting data from a single fly, one at a time, and enabled the data acquisition process to be more efficient. In addition, the automated RING assay has been employed in a number of studies to elucidate the mechanism underlying Alzheimer&#39;s and Parkinson&#39;s Disease, validating the approach with high efficiency7,8,9.
In this article, we demonstrate the automated RING assay using the DDC-Gal4 driven RNAi flies. DDC-Gal4 is a Gal4 line specifically expressing in dopaminergic (DA) and serotonergic neurons, thus representing a great tool for analyzing the target gene effects associated with locomotor deficits accompanying neurodegeneration10. In addition, we incorporate UAS-Dicer2, a fly line that enhances RNAi efficiency, to generate the UAS-Dicer2; DDC-Gal4 tool line. The RNAi flies we choose to use is the auxilin (aux) RNAi v16182 (auxR16182), a gene that we have previously identified to exhibit an effect on fly locomotor activity8. auxGFP flies are also prepared for analyzing effects upon aux overexpression. We will show how to use the automated RING assay to measure fly negative geotaxis, present the results, and discuss any implications acquired from the results.

<table>
	<tbody>
		<tr>
			<td>Forma Environmental Chamber</td>
			<td>Thermo&#160;</td>
			<td>3949</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon dioxide cylinders</td>
			<td>FuLian GAS Technology</td>
			<td>GB/T6052</td>
			<td></td>
		</tr>
		<tr>
			<td>HDR-Camcorder</td>
			<td>SONY</td>
			<td>HDR-CX220E</td>
			<td></td>
		</tr>
		<tr>
			<td>Binocular stereomicroscope</td>
			<td>Xin Zhen</td>
			<td>SMZ-168BL</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic scales</td>
			<td>MinQiao</td>
			<td>SL1002N</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerator</td>
			<td>Haier</td>
			<td>SC-350</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar-agar powder</td>
			<td>Sinopharm</td>
			<td>10000561</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sinopharm</td>
			<td>10010518</td>
			<td></td>
		</tr>
		<tr>
			<td>Corn meal</td>
			<td>Sinopharm</td>
			<td>5464654</td>
			<td></td>
		</tr>
		<tr>
			<td>Brown sugar</td>
			<td>LiuCaiYuan</td>
			<td>45467936</td>
			<td></td>
		</tr>
		<tr>
			<td>Instant dry yeast</td>
			<td>AB MAURI</td>
			<td>20886</td>
			<td></td>
		</tr>
		<tr>
			<td>AuxR16182</td>
			<td>VDRC</td>
			<td>7187</td>
			<td></td>
		</tr>
		<tr>
			<td>UAS-Dicer2</td>
			<td>Bloomington</td>
			<td>24650</td>
			<td></td>
		</tr>
		<tr>
			<td>UAS-mCD8GFP</td>
			<td>Bloomington</td>
			<td>32185</td>
			<td></td>
		</tr>
		<tr>
			<td>DDC-Gal4</td>
			<td></td>
			<td></td>
			<td>A gift from Fude Huang</td>
		</tr>
		<tr>
			<td>AuxGFP</td>
			<td></td>
			<td></td>
			<td>A gift from Henry Chang</td>
		</tr>
	</tbody>
</table>

an automated rapid iterative negative geotaxis assay for analyzing adult climbing behavior in a drosophila model of neurodegeneration

Neurodegenerative diseases are frequently associated with a progressive loss of movement ability, reduced life span, and age-dependent neurodegeneration. To understand the mechanism of these cellular events, and their causal relationships with each other, Drosophila melanogaster, with its sophisticated genetic tools and diverse behavioral features, are used as disease models for assessing neurodegenerative phenotypes. Here we describe a high-throughput method to analyze Drosophila adult negative geotaxis behavior, as an indication for possible motor defects associated with neurodegeneration. An automated machine is designed and developed to drive fly synchronization using an initial electric impulse, later allowing the recording of negative geotaxis behavior over a course of secs to mins. Images from the digitally recorded video are then processed with the self-designed RflyDetection software for statistical data manipulation. Different from the manually controlled negative geotaxis assay based on single fly, this precise, fast, and high-throughput protocol allows data acquisition from more than hundreds of flies simultaneously, providing an efficient approach to advance our understanding in the underlying mechanism of locomotor deficits associated with neurodegeneration.

This article demonstrates the use of an automated RING assay in assessing fly negative geotaxis behavior. Unlike the previous RING assay, our assay includes an automated apparatus that provides an electric force to synchronize fly action and analyzes up to hundreds of flies simultaneously (Figure 1). Analysis of Dicer2; DDC&#62;auxR16182 flies showed an age-dependent decrease in the climbing distance in a 6-s time frame for both male and f...

Watch this Scientific Journal Video about An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration at JoVE.com

An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration

This step-by-step protocol analyzes Drosophila negative geotaxis behavior using an automated multi-cylinder system that hosts hundreds of flies and synchronizes their action by an electric motor. Upon synchronization, fly negative geotaxis behavior is assayed, digitally recorded, and analyzed using the self-designed RflyDetection software.

An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration

Neurodegenerative diseases are frequently associated with a progressive loss of movement ability, reduced life span, and age-dependent neurodegeneration. ...

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