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. ...

The overall goal of this automated rapid iterative negative geotaxis assay is to analyze Drosophila adult climbing deficits associated with neurodegeneration. This method can help to answer key questions in the neuroscience field such as the mechanism of locomotive deficits associated with neurodegeneration. The main advantage of this technique is that fly climbing activity can be analyzed in an automated and high proof profession.The implications of this technique extends towards the therapy and diagnosis of neurodegeneration, because neurodegenerative diseases are frequently associated with a progressive loss of movement ability and reduced lifespan and age dependent neurodegeneration. We first had the idea for this method. We wanted to measure Drosophila adult's locomotive activity in a more accurate fashion.To carry out fly collection, begin by maintaining the flies on standard fly food at 25 degrees Celsius, 70%humidity and to 12 hour light, 12 hour dark cycle. Collect UAS-Dicer2 DDC-GAL4 fly virgins under carbon dioxide anesthesia. Cross the virgins to two-day old adult male flies carrying the following genotypes, with a male/female ratio of one-to-two.Separately collect newly occlosed males and females in three vials for each group per experiment, placing 10 flies in each regular fly vial with standard food. Then set the vials to 25 degrees Celsius. To perform the automated RING assay, transfer 10 collected unisex flies per genotype into each vial.Then secure the vial with a screw. Analyze control flies and flies carrying different genotypes together for each set of experiments. Turn on the digital camera placed in front of the apparatus, and once the flies are loaded and ready, start recording.The camera should be placed horizontally and the camera a proper distance from the RING apparatus to capture the positions in a relatively accurate locations of all the flies. After allowing one minute for the flies to settle in the vials, turn on the step controller that controls the step driver. This drives the small electric motor to control the attached lever, so that it consecutively rises and taps the apparatus four times in two seconds.After tapping the apparatus, note that the flies begin to ascend the wall. With recording still on, repeat the synchronization as just demonstrated, in 60 second intervals for three to five consecutive trials. Repeat the experiment for five, 15, 25 and 35 day old flies.Conduct at least three independent experiments for each group for each experiment, with at least 30 collected flies or three vials. To analyze the data, import the recorded video into the computer. For each trial, take a snapshot of the video at six seconds after tapping.In the R-fly detection software, use the file menu to import the snapshot image. Next, using the baseline icon on the Toolbar, precisely set the upper and lower baselines of the vial and then use the cursor to mark the upper and lower baselines on the image. Within the Settings bar, in the Flies in Rect field, input the number of flies per vial, and in the Tube Height field, input the vial length.Individual fly positions are detected and labeled with dots on the screen for each vial. Note that in a table on the right hand panel, the software automatically determines the climbing distance for each fly and displays the averaged values from 10 flies in each vial. Present the data as the mean plus or minus SEM.Calculate the P-values of significance using a one-way ANOVA with a Bonferroni multiple comparison test. Analysis of Dicer2 designated flies using the automated RING assay presented in this video showed an age dependent decrease in the climbing distance in a six-second timeframe for both male and female flies, suggesting that aux expression in dopaminergic or DA neurons is crucial for fly locomotor activity. Climbing ability does not seem to be affected upon auxGFP over expression in DA neurons and co-expression of auxR and auxGFP in DA neurons rescued from the RNAi phenotype.Once mastered, this can be done is two to three hours if it is performed properly. Having this development is now a way for researchers in the field of neuroscience to explore neurodegeneration in Drosophila melanogaster. Don't forget that working with electric RING apparatus can be extremely hazardous and each confirm should be fastened tightly and carefully checked before performing this procedure.

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9.6K visualizações.  Tongji University School of Medicine. The overall goal of this automated rapid iterative negative geotaxis assay is to analyze Drosophila adult climbing deficits associated with neurodegeneration. This method can help to answer key questions in the neuroscience field such as the mechanism of locomotive deficits associated with neurodegeneration. The main advantage of this technique is that fly climbing activity can be analyzed in an automated and high proof profession.The implications of this technique extends towards the ...