Name: Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration (Video) | JoVE Journal
Uploaded: 2011-03-11T13:00:00.000+00:00
Duration: 7 min 25 s
Description: Learn behavioral assays for locomotor, learning, and memory deficits in Drosophila models of neurodegeneration. This method uses negative geotaxis and aversive phototaxis suppression to evaluate cognitive functions.

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The authors would like to thank Dr. Charles Luetje for providing the T-maze and the behavior room. This work is supported by the American Heart Association (to Y.A.), the National Institute for Neurological Disorder and Stroke Grant R01NS64269 (to R.G.Z.), and the Pew Charitable Trust (to R.G.Z.).

1. 	Introduction
In this video, we will demonstrate two behavioral assays using Drosophila to demonstrate the measurement of locomotion and learning and memory functions that are compromised in neurodegenerative disorders. First, we will show how locomotor behavior can be measured using the negative geotaxis assay. Second, we will assess learning and memory abilities using positively-phototactic flies that are trained to associate light with an aversive bitter taste and then tested 6 hours later to assess memory functions. Finally, we will discuss the implications and limitations of these behavior assays.
2. 	Equipment and Reagents
<ol>
<li>Fly work
<ul>
<li>Stereo scope (Zeiss)</li>
<li>Small paint brushes for pushing flies</li>
<li>Drosophila carbon dioxide (CO2) anesthesia apparatus (Geneseesci, Inc)</li>
</ul>
</li>
<li>Negative Geotaxis Assay
<ul>
<li>28.5 x 95 mm Polystyrene Vial (Capitol Vial, Inc.)</li>
<li>Mini-Alarm Timer/Stopwatch (VWR International) </li>
<li>Sharpie pens</li>
<li>Adhesive Tape</li>
</ul>
</li>
<li>Aversive Phototaxis Suppression Assay
<ul>
<li>Quinine Hydrochloride (Sigma Aldrich; CAS Number: 6119-47-7)</li>
<li>Distilled Water</li>
<li>Digital measuring scale for precise dilution by weight</li>
<li>Quick Disconnect Connectors, High-Density Polyethylene (NALGENE VWR Catalog No. 62868-021)</li>
<li>15 mL Centrifuge Tube (VWR International), cut at the 2 mL mark from closed end to fit with the Quick Disconnect Connector adaptor for light source </li>
<li>Single-channel 200 μL pipetter to load quinine solution onto filter paper lining the chamber</li>
<li>T-Maze (Simple Behavioral Systems; Figure 1)</li>
<li>Aluminum Foil</li>
<li>Light Source (Zeiss)</li>
<li>A lamp with red light.</li>
<li>Filter Paper</li>
<li>Mini-Alarm Timer/Stopwatch (VWR International)</li>
</ul>
</li>
</ol>
3. 	Preparing the Flies
<ul>
<li>Flies are maintained on standard cornmeal-agar-molasses-yeast medium at 25°C on a 12 hr light/dark cycle. </li>
<li>Virgin flies are isolated under carbon dioxide anesthesia on the day of eclosion. For aging studies, flies are maintained in groups of twenty per vial and transferred to a fresh vial every 3 days. It is important to use sibling flies from the same parental cross for aging studies, to minimize differences arising from genetic backgrounds.</li>
<li>For both the assays, flies are sexed and maintained in groups of ten flies per vial. The gender difference on behavior is significant and it is advisable not to mix male and female flies in a study as data might be hard to interpret and reproduce6. </li>
<li>For negative geotaxis assay, flies are sorted into groups of ten per vial and tested one hour after anesthesia. It is important for flies to recover completely from anesthesia as this might have an effect on locomotor activities.</li>
<li>For APS assay, flies of each group are placed in an empty polystyrene vial with water moistened filter paper for six hours before the assay is performed. This ensures that the flies are starved before the assay and will be more perceptive to aversive taste. Just before the assay is performed, each fly from a group of ten is placed in an empty 15mL centrifuge tube, which is then capped loosely, to avoid escape. </li>
</ul> 
4. 	Negative Geotaxis Assay
<ul>
<li>Geotaxis is generally measured for ten to twenty groups of ten individuals of the same genotype or treatment (100-200 flies total for each genotype/treatment)7. Sort groups of ten female or male flies on a CO2 anesthesia apparatus and place each group in a separate vial. Allow at least one hour for the flies to recover from anesthesia.</li>
<li>Prepare the climbing apparatus for each group, such that two empty polystyrene vials are vertically joined by tape facing each other. It is important to make sure that the openings of the vials are perfectly aligned with each other to provide an even climbing surface for the flies. </li>
<li>For the lower vial, measure a vertical distance of 8 cm above the bottom surface and mark each vial by drawing a circle around the entire circumference of the vial.</li>
<li>Transfer a group of ten flies into the lower vial carefully preventing the escape of any fly. Immediately cover the lower vial with the top vial and tape securely near the contacting openings. Allow the flies to acclimatize to the new setting for 1 minute before conducting the assay.</li>
<li>Gently tap the flies down to the bottom of the vial and measure the number of flies that can climb above the 8-cm mark by 10 seconds after the tap.</li>
<li>Repeat this assay for the same group ten times, allowing for 1 minute rest period between each trial.</li>
<li>Record the number of flies per group that passed the 8-cm mark as a percentage of total flies. </li>
</ul>
5. 	Aversive Phototaxis Suppression Assay (APS Assay)
Originally adapted from Le Bourg and Buecher (2002)4, this assay exploits the positive phototactic behavior in flies to train them to associate light with aversive stimuli (in this case, bitter taste of quinine). After a training/conditioning phase, wild type flies will be able to associate the lighted area with aversive taste and avoid it. Flies with compromised learning capacities will fail to make this association. Furthermore, the APS assay can also be used to measure short term memory function of flies5 by subjecting already trained flies to the same test 6 hours post conditioning to test their ability in remembering the learned task.
<ol>
<li>Preparing the Quinine Solution
<ul>
<li>Dissolve quinine hydrochloride in distilled water to make a 0.1M stock solution (1.98g in 50mL distilled water). The stock solution can be kept at -20°C for up to a year in small aliquots</li>
<li>Prepare a working solution of 1 μM by diluting the stock solution in distilled water.</li>
</ul>
</li>
<li>Preparing the T-Maze
<ul>
<li>T-maze consists of the center column with the trap door and two independent chambers, a "dark" chamber and a "lighted" chamber (Figure 1). </li>
<li>Chamber preparation: take two 15mL plastic centrifuge tubes and cut at 2 mL mark from the bottom closed end with a manual saw. Fit the connector adapters at each end, and seal the connection with parafilm. The adapter at the end of the tube serves as the input slot for the gooseneck light source. Connect one tube with the light source and this tube will be the "lighted" chamber. Wrap the other tube with aluminum foil to serve as the "dark" chamber. </li>
<li>Add 180uL of either distilled water or quinine solution to filter paper and place in the light chamber.</li>
<li>Assemble the T-maze by screwing the lighted and dark chambers on each side of the center column, with the trap door in the middle closed (Figure 1). </li>
<li>Insert the light source into the lighted chamber. </li>
</ul>
</li>
<li>Training flies to suppress phototaxis with aversive stimuli
<ul>
<li>Unscrew the dark chamber from the T-maze and transfer a single fly into this chamber and immediately screw the dark tube back to the maze.</li>
<li>Turn off the lights in the room and turn on the red lamp.</li>
<li>Allow the fly to acclimatize in the dark chamber for 30 seconds and slowly turn on the light source illuminating the lighted chamber. Slowly open the trap door that separates the two chambers.</li>
<li>If the fly walks to the lighted chamber within 10 seconds, it is considered positively phototactic and is ready to be trained for the assay. Failure in phototaxis indicates problems in the visual system and flies with negative phototaxis need to be excluded from the assay. </li>
<li>For Aversive Phototaxis Suppression (APS) training, tap the fly that demonstrated positive phototaxis back to the dark chamber, close the trap door and turn off the light. Allow 30 seconds of acclimatizing. During this time put filter paper with quinine solution into the lighted chamber. Slowly open the trap door and turn on the light. Allow the fly walk into the quinine coated lighted chamber. After one minute, tap the fly back to the dark chamber and repeat this 9 more times. (Typically, wild type flies avoid the lighted chamber after 3 to 5 training trials.)</li>
<li>Immediately after training, five test trials will be conducted. In each test trial, allow 10 seconds after light is turned on for the trained fly to walk to the lighted chamber. Failure to walk to the lighted vial will be recorded as "Pass", which is equivalent to "task learned through reinforcement". The Pass rate over five consecutive trials is recorded and shown below in Figure 2 as PC0 (0 hr post conditioning).</li>
</ul>
</li>
<li>Assessing Short-Term Memory Function in Flies
<ul>
<li>After training and initial PC0 recordings, each fly is placed back into its original food vial and kept aside for six hours.</li>
<li>Six hours post training, subject each fly to 5 trials again in the same way as before, and record the number of times the fly avoids (pass) or goes into (fail) the lighted vial. This pass rate is recorded as PC6 (6 hrs post conditioning), which is an indicator of short term memory.</li>
</ul>
</li>
</ol>
6. 	Data Interpretation and Statistical Analysis
<ol>
<li>Negative Geotaxis Assay
<ul>
<li>The raw data generated from this assay represents the number of flies crossing the 8-cm mark in 10 seconds in each group. Convert this to percentage and compute the average pass rate for each group over 10 sessions.</li>
<li>Data is represented graphically as an average pass rate per group (genotype, treatment etc.) with the standard error of mean (SEM).</li>
<li>Differences between groups can then be determined to be statistically significant or not using a Student T-test or ANOVA.</li>
</ul>
</li>
<li>Aversive Phototaxis Suppression Assay
<ul>
<li>The raw data from this assay is represented as the number of times each fly avoids the lighted chamber in five successive trials. The pass rate is calculated as the percentage of successful avoidance trials over the total trials. Calculate the average pass rate in this manner for a minimum of 15 flies per group.</li>
<li>Data is graphically represented as average pass rate PC0 (immediately after conditioning; "learning" indicator) or average pass rate PC6 (6 hours post conditioning; "memory" indicator) with respective SEM.</li>
<li>Learning and memory indices are compared between groups with both wild type and "genetically-manipulated-benign" group (such as GFP overexpressing flies) appropriately.</li>
</ul>
</li>
</ol>
7. 	Representative Results
In this video experiment, we are assessing learning and memory and motor deficits in flies overexpressing human Tau in the central nervous system and comparing their performance to those of flies overexpressing GFP. Neuronal overexpression of human Tau in Drosophila has been shown to cause severe vacuolization in the brain8 and lead to significant deficits in learning and memory functions9. As shown in Figure 2A, 10 day old male or female flies overexpressing human Tau have significant locomotor deficits compared to age-matched GFP overexpressing flies. In addition, these flies failed to associate light with bitter taste as shown by PC0 pass rate compared to the control group, as well as fail to remember this association as shown by a decline in PC6 pass rate (Figure 2B). This experiment further validates that Drosophila models can be used for studying human neurodegenerative conditions and recapitulating some of the behavioral phenotypes observed in humans and mammals.
<img src="/files/ftp_upload/2504/2504fig1.jpg" alt="Maze apparatus with quinine filter for light/dark training setup in behavioral experiment." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1. T-Maze Setup for Aversive Phototaxis Suppression Assay. A. The overall experimental setup for the APSA experiment with the light source connected to the "lighted" falcon tube, lined with filter paper and the "dark" tube on the left (covered in foil), separated by a trap door. B. During the training and testing phase, the fly is in the dark chamber and the trap door opened after the light is turned on in the lighted chamber.
<img src="/files/ftp_upload/2504/2504fig2.jpg" alt="Climbing pass rate analysis bar graph, 10-20 days post eclosion, comparing genetic variants." data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2. Representative results from the negative geotaxis and APS experiment using flies overexpressing GFP or Tau with a pan-neuronal driver. A. In the negative geotaxis assay, 10-day old male (blue column) or female (red column) flies overexpressing GFP showed higher climbing activity than Tau-overexpressing flies. B. 20-day old female flies overexpressing GFP showed better learning (PC0) and memory (PC6) functions than Tau overexpressing flies.

<ol><li>Bonini, N. M., Fortini, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+neurodegenerative+disease+modeling+using+Drosophila%5BTitle%5D)+AND+%22Annual+Review+of+Neuroscience%22%5BJournal%5D)">Human neurodegenerative disease modeling using Drosophila.</a> Annual Review of Neuroscience. 26, 627-656 (2003).</li>
<li>Lloyd, T. E. &amp, Taylor, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flightless+flies%3A+Drosophila+models+of+neuromuscular+disease%5BTitle%5D)+AND+%22Ann+N+Y+Acad+Sci%22%5BJournal%5D)">Flightless flies: Drosophila models of neuromuscular disease.</a> Ann N Y Acad Sci. 1184, e1-e20 (2010).</li>
<li>Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Behavioral+mutants+of+Drosophila+isolated+by+countercurrent+distribution%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+USA%22%5BJournal%5D)">Behavioral mutants of Drosophila isolated by countercurrent distribution.</a> Proc. Natl. Acad. Sci. USA. 58, 1112-1119 (1967).</li>
<li>Le Bourg, E., Buecher, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Learned+suppression+of+photopositive+tendencies+in+Drosophila+melanogaster%5BTitle%5D)+AND+%22Animal+Learning+%26+Behavior%22%5BJournal%5D)">Learned suppression of photopositive tendencies in Drosophila melanogaster.</a> Animal Learning & Behavior. 30, 330-341 (2002).</li>
<li>Seugnet, L., Suzuki, Y., Stidd, R., Shaw, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aversive+phototaxic+suppression%3A+evaluation+of+a+short-term+memory+assay+in+Drosophila+melanogaster%5BTitle%5D)+AND+%22Genes+Brain+Behav%22%5BJournal%5D)">Aversive phototaxic suppression: evaluation of a short-term memory assay in Drosophila melanogaster.</a> Genes Brain Behav. 8, 377-389 (2009).</li>
<li>Nilsen, S. P., Chan, Y. -B., Huber, R., Kravitz, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gender-selective+patterns+of+aggressive+behavior+in+Drosophila+melanogaster%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+USA%22%5BJournal%5D)">Gender-selective patterns of aggressive behavior in Drosophila melanogaster.</a> Proc. Natl. Acad. Sci. USA. 101, 12342-12347 (2004).</li>
<li>Leal, S. M., Neckameyer, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pharmacological+evidence+for+GABAergic+regulation+of+specific+behaviors+in+Drosophila+melanogaster%5BTitle%5D)+AND+%22J+Neurobiol%22%5BJournal%5D)">Pharmacological evidence for GABAergic regulation of specific behaviors in Drosophila melanogaster.</a> J Neurobiol. 50, 245-261 (2002).</li>
<li>Wittmann, C. W., Wszolek, M. F., Shulman, J. M., Salvaterra, P. M., Lewis, J., Hutton, M., Feany, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tauopathy+in+Drosophila%3A+Neurodegeneration+Without+Neurofibrillary+Tangles%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Tauopathy in Drosophila: Neurodegeneration Without Neurofibrillary Tangles.</a> Science. 293, 711-714 (2001).</li>
<li>Mershin, A., Pavlopoulos, E., Fitch, O., Braden, B. C., Nanopoulos, D. V., Skoulakis, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Learning+and+memory+deficits+upon+TAU+accumulation+in+Drosophila+mushroom+body+neurons%5BTitle%5D)+AND+%22Learn+Mem%22%5BJournal%5D)">Learning and memory deficits upon TAU accumulation in Drosophila mushroom body neurons.</a> Learn Mem. 11, 277-287 (2004).</li>
<li>Ganetzky, B., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Indirect+Suppression+Involving+Behavioral+Mutants+with+Altered+Nerve+Excitability+in+DROSOPHILA+MELANOGASTER%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Indirect Suppression Involving Behavioral Mutants with Altered Nerve Excitability in DROSOPHILA MELANOGASTER.</a> Genetics. 100, 597-614 (1982).</li>
<li>Hall, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Acetylcholinesterase+mutants+in+Drosophila+and+their+effects+on+the+structure+and+function+of+the+central+nervous+system%5BTitle%5D)+AND+%22J+Comp+Neurol%22%5BJournal%5D)">Acetylcholinesterase mutants in Drosophila and their effects on the structure and function of the central nervous system.</a> J Comp Neurol. 189, 741-774 (1980).</li>
</ol>

No conflicts of interest declared.

Both negative geotaxis and APS assays are robust behavioral assays to measure changes in locomotor or learning and memory capacities arising from genetic or environmental manipulation. However, there are some disadvantages to these assays that await further optimization.
In negative geotaxis assay, mutations in certain genes can render severe motor dysfunctions in flies. In such cases, the flies can hardly climb the walls in response to gravity upon agitation. Other behavior assays that directly measure movement may be more appropriate. These assays include righting reflex behavior that measures paralysis and seizure10, and optomotor behavior11 that records loss of movement coordination.
In APS assay, the first hindrance can be flies randomly stopping at the entrance of the lighted chamber without moving forward. This becomes more of an issue if aging is one of the conditions studied, since older flies are less active in general and tend to stop and rest for long periods of time. In this case the experimenter has to provide mechanical stimuli to promote movement, which is not always effective. Second, one might argue that the failure to go to the lighted chamber results from a loss of positive phototaxis. This is rarely the case, since previous work has shown that flies turn back to light responsiveness once the negative reinforce is removed4. Third, the duration of contact with the aversive stimulus may vary from trial to trial. This variation can usually be reduced by encircling the inner wall of the chamber with soaked filter paper to prevent flies from avoiding the quinine. In addition, the experimenter can ensure that each fly spend enough time in contact with the filter paper by increasing the amount of time spent in the chamber from the otherwise noted 1 minute. 
In summary, negative geotaxis and APS assays are fast and simple ways of assessing behavioral deficits and quantitatively indicate locomotor and learning and memory abilities in Drosophila. These assays are particularly useful in modeling human neurodegenerative conditions in Drosophila.

<table><tbody><tr><td> Polystyrene Vial </td><td>Capitol Vial, Inc.</td><td>28Fly</td><td> 28.5 x 95 mm Polystyrene Vial; material quality important, bad if too slippery for flies to climb.</td></tr><tr><td> Dissecting Stereoscope</td><td>Carl Zeiss, Inc.</td><td>000000-1432-979</td><td> 2 branches goose-neck light guide (12V/30W) </td></tr><tr><td> Quinine Hydrochloride</td><td>Sigma-Aldrich</td><td>6119-47-7</td><td> Dissolve in distilled water.</td></tr><tr><td> Quick Disconnect Connectors </td><td>Nalge Nunc international</td><td>62868-021</td><td> High-Density Polyethylene</td></tr><tr><td> T-Maze </td><td>Simple Behavioral Systems</td><td></td><td></td></tr><tr><td> Single-channel 200 &amp;mu;L pipette </td><td>Eppendorf</td><td>022443305</td><td></td></tr></tbody></table>

assaying locomotor, learning, and memory deficits in drosophila models of neurodegeneration

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of these diseases, including Alzheimer's, Parkinson's and Huntington's disease are characterized by age-dependent deterioration in learning and memory functions and movement coordination2. Here we use behavioral assays, including the negative geotaxis assay3 and the aversive phototaxic suppression assay (APS assay)4,5, to show that some of the behavior characteristics associated with human neurodegeneration can be recapitulated in flies. In the negative geotaxis assay, the natural tendency of flies to move against gravity when agitated is utilized to study genes or conditions that may hinder locomotor capacities. In the APS assay, the learning and memory functions are tested in positively-phototactic flies trained to associate light with aversive bitter taste and hence avoid this otherwise natural tendency to move toward light. Testing these trained flies 6 hours post-training is used to assess memory functions. Using these assays, the contribution of any genetic or environmental factors toward developing neurodegeneration can be easily studied in flies.

Behavior assays for measuring locomotor functions, learning, and memory abilities in Drosophila.

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of ...

The overall goal of the procedure is to study learning and memory in adult Esso Melan Gaster first to determine if the flies suffer from locomotor deficits. They are tested in the negative geo taxes. Assay positive photo tactic behavior is then verified by placing flies in a T maze and recording the latency to move toward the favored lighted chamber.Flies with positive phototaxis are then conditioned to associate the lighted chamber with the bitter taste of quinine. Ultimately, flies are tested on how well they associate the aversive stimulus with the lighted chamber providing a measure of learning and memory. Ola, welcome to the X lab.Today we'll be demonstrating to you two commonly used behavioral assays to study neurodegeneration and drosophila. First, we'll show you the aversive phototaxis suppression assay demonstrated to you by Wil Fredo sala, an undergraduate in our lab and myself, which will be followed by the negative geo taxes assay demonstrated to you by kaiwan, a graduate student in our lab. Maintain the flies on the standard cornmeal agar molasses yeast medium at 25 degrees Celsius on a 12 hour light dark cycle.On the day of the occlusion, sort virgin flies into groups of 20. Virgin flies are identified by their light color and a dark greenish spot located on the underside of their abdomen. The flies should be transferred into a fresh vial every three days, and sibling flies should be used to minimize differences arising from genetic backgrounds.10 to 20 groups per genotype or treatment should be tested in a geo taxes study. To begin sort female and male flies into groups of 10. These flies should be tested separately.Since behavior is significantly influenced by gender. Wait at least one hour after anesthesia before proceeding to ensure that locomotor activity is unimpaired. Once the flies are prepared perfectly, align the openings of two empty polystyrene vials by joining them vertically on the lower vial.Measure eight centimeters from the bottom and draw a circle around the circumference. Now that the apparatus has been assembled, transfer a group of 10 flies into the lower vial. Immediately rejoin the vials and acclimate the flies for one minute before testing.After the flies have settled, gently tap them down to the bottom of the vial and measure the number of flies that climb above the eight centimeter mark by 10 seconds. The number of flies per group that passed this mark is recorded as a percentage of total flies. Repeat this procedure 10 times per group, allowing the flies to rest for one minute between trials.Six hours before the a PS assay, sort, female flies into polystyrene vials containing water moistened filter paper. This step ensures that the flies are starved and more perceptive to the aversive taste. In this assay, a quinine solution serves as the aversive stimulus.Prepare a working solution of one micromolar from a 0.1 molar stock. The T maze consists of a center column with a trap door and two side chambers, a dark chamber and a lighted chamber. At the two millimeter mark, use a manual saw to cut off the closed end of two 15 milliliter plastic centrifuge tubes fit connector adapters at each end and seal the connection with paraform.Wrap one tube with aluminum foil to service the dark chamber. Assemble the TMAs by screwing the lighted and dark chambers on each side of the center column. With the trap door in the middle closed, complete the setup by connecting a light source to the adapter of the light chamber directly Before performing the assay transfer each fly from a group of 10 into an empty 15 milliliter centrifuge tube and cap lightly to prevent escape.Next, unscrew the dark chamber from the TMAs. Transfer a single fly into the tube and immediately insert it back into the maze. Turn off the lights in the room and turn on a red lamp.Allow the fly to acclimatize in the dark chamber for 30 seconds and then slowly turn on the light source illuminating the lighted chamber. Once lighted, open the trap door that separates the two chambers. If the fly walks to the lighted chamber within 10 seconds, it is considered positively photo tactic and is ready to be trained.Failure to respond in this way indicates a problem in the visual system and necessitates exclusion from the assay. Once positive phototaxis has been confirmed, tap the fly back to the dark chamber, close the trap door and turn off the light during a 32nd rest period. Prepare the aversive stimulus by placing the filter paper into the lighted chamber and pipetting one milliliter of quinine solution onto the filter paper.Ensuring all of the filter paper is completely moist with aversive stimuli. To begin conditioning slowly open the trap door and turn on the light. Allow the fly to walk into the quinine scented lighted chamber.After one minute, tap the fly back to the dark chamber. Repeat this procedure nine times for a total of 10 conditioning trials. Typically while type flies, avoid the lighted chamber after three to five training trials immediately after training.Conduct five test trials. If the trained fly does not walk into the light of chamber within 10 seconds, it is recorded as a pass. The pass rate over five consecutive trials is recorded and designated.PC zero. After training and initial PC zero recordings, each fly is placed back into its original numbered food file and kept aside for six hours. Six hours after training, conduct five additional test trials as demonstrated earlier, this pass rate represents PC six and is a measure of short-term memory.In the negative geo taxes assay, the number of flies crossing the eight centimeter mark is converted to a percentage and presented as the climbing pass rate. Over 10 trials as seen here on the right, 10 day old male or female flies over expressing. Human tau have significant locomotor deficits compared to age match GFP over expressing flies on the left.Performance in the aversive phototaxis suppression assay is represented by the average pass rate or the percentage of successful avoidance trials. Over total trials flies over expressing tau shown here on the right pale to associate the light with a bitter taste when compared to the control group on the left, both immediately after training and when tested, six hours later Following this procedure, samples can be taken for immunohistochemical analysis to answer additional question about morphological differences. After watching this video, you should have a good understanding of how to study learning and memory and flies amigos.And remember, it's all about the you.

assaying-locomotor-learning-memory-deficits-drosophila-models

Research

JoVE Journal

Neuroscience

The method to measure adult Drosophila associative memory is described. The assay is based on the ability of the fly to associate an odor presented with a negative reinforcer (electric shock) and then recall this information at a later time, allowing memory to be measured.

Drosophila Adult Olfactory Shock Learning

Here we describe two assays that have been established to study age-dependent neurodegeneration of dopaminergic (DA) neurons in Drosophila: the climbing/startle-induced negative geotaxis assay which allows to study the functional effects of DA neurons degeneration and the tyrosine hydroxylase immunostaining which is used to identify and count DA neurons in whole brain mounts.

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets.

Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

Drosophila is widely used as a model system to study neurodegeneration. This protocol describes a method by which degeneration, as determined by vacuole formation in the brain, can be quantified. It also minimizes effects due to the experimental procedure by processing and sectioning control and experimental flies as one sample.

Mass Histology to Quantify Neurodegeneration in Drosophila

This protocol describes a Drosophila learning and memory assay called courtship conditioning. This classic assay is based on a reduction of male courtship behavior after sexual rejection by a non-receptive premated female. This natural form of behavioral plasticity can be used to test learning, short-term memory, and long-term memory.

Drosophila Courtship Conditioning As a Measure of Learning and Memory

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

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

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

The goal of this procedure is to dissect the dorsal longitudinal muscle (DLM) tissue to assess the structural integrity of DLM neuromuscular junctions (NMJs) in neurodegenerative disease models using Drosophila melanogaster.

Visualizing Synaptic Degeneration in Adult Drosophila in Association with Neurodegeneration

The present protocol assesses the locomotor activity of Drosophila by tracking and analyzing the movement of flies in a hand-made arena using open-source software Fiji, compatible with plugins to segment pixels of each frame based on high-definition video recording to calculate parameters of speed, distance, etc.

A Simple Technique to Assay Locomotor Activity in Drosophila

The protocol presents a high-throughput, cost-effective method for assessing neurotoxicity in the fruit fly through the quantification of locomotor dysfunction as an alternative to traditional mammalian models. It aims to evaluate the neurotoxic effects of pharmaceutical compounds, environmental agents, or genetic modifications using a sensitive, reproducible, and ethically favorable model.

Behavioral Analysis of Locomotor Dysfunction in Drosophila melanogaster as a Readout for Neurotoxicity

Learn behavioral assays for locomotor, learning, and memory deficits in Drosophila models of neurodegeneration. This method uses negative geotaxis and aversive phototaxis suppression to evaluate cognitive functions.

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

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