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

Summary

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.

Abstract

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and thus current therapeutic treatment strategies are not curative. Through the use of whole exome sequencing, an increasing number of genes have been identified to play a role in human locomotion. Despite identifying these genes, it is not known how these genes are crucial to normal locomotive functioning. Therefore, a reliable assay, which utilizes model organisms to elucidate the role of these genes in order to identify novel targets of therapeutic interest, is needed more than ever. We have designed a sensitized version of the negative geotaxis assay that allows for the detection of milder defects earlier and has the ability to evaluate these defects over time. The assay is performed in a glass graduated cylinder, which is sealed with a wax barrier film. By increasing the threshold distance to be climbed to 17.5 cm and increasing the experiment duration to 2 min we have observed a greater sensitivity in detecting mild mobility dysfunctions. The assay is cost effective and does not require extensive training to obtain highly reproducible results. This makes it an excellent technique for screening candidate drugs in Drosophila mutants with locomotion defects.

Introduction

Devastating neurodegenerative disorders such as Parkinson’s disease, amyotrophic lateral sclerosis, and hereditary spastic paraplegia are increasingly recognized. Unfortunately, most of these neurodegenerative disorders are still without treatments. The widespread clinical use of genome-wide, unbiased genetic tests such as whole exome sequencing has led to an increasing number of genes being implicated in human locomotive disorders. Despite this progress, the pathological progression from early to late stages, remains elusive in these disorders. Drosophila provides one with the genetic tools for studying gene requirement in a controlled spatial and temporal manner. In addition, Drosophila has proven useful in screening drugs for neurological conditions such as Parkinson’s¹, Alzheimer’s², intellectual disability^3,4 and epilepsy^5,6 among others. Our aim was to develop a cost effective and reliable assay that would allow high throughput analysis that would still be sensitive enough to detect small changes in motor performance.

There are several assays used to quantify the effects of genetic mutation and/or environmental condition on Drosophila climbing behavior. Most of the assays capitalize on the natural tendency of flies to climb, known as negative geotaxis, or the climbing assay. Benzer⁷ suggested in 1967 that the counter-current apparatus used for the study of phototaxis could also be used to study gravitaxis. Since then, Ganetsky⁸ and many others^9-12 have built on the initial assay. The principle is to place a known number of flies in a vial and tap the vial strongly against a hard surface, causing the flies to fall to the bottom of the vial. As it is an innate behavior, the flies will attempt to climb to the top of the vial, opposed to gravity. This assay is quantitative and measures how many flies have climbed past a marker on the vial during an allotted time period. Measurement of speed instead of total number of flies climbing has become a reliable parameter and shown defects in cases where the number of flies criteria was not significant¹³.

The climbing assay has proven useful in the study of many neurodegenerative disorders including Parkinson’s disease¹⁴. However, we noted that locomotive defects may not be detectable at time where neurodegeneration is already seen in pathological studies¹⁴. Thus, use of the traditional assay may limit the ability to study the early stages of disease pathogenesis. The appearance of locomotive defects during later stages of pathology may reflect a disease whose progression is too advanced for complete rescue.

This raises a potential issue with the sensitivity of the traditional climbing assay. The potential inability of the traditional climbing assay to detect mild locomotive defects can be attributed to the height to which the flies are required to climb. The traditional assay^15,16 measures the number of flies to successfully climb over a height of 2 to 5 cm in 10 to 20 sec.

Protocol

Research on Drosophila melanogaster was in compliance with the University of Alberta’s research guidelines.

1. Fly Collection

1. Collect 20 flies using CO_{2 (g)} anesthetization and place in a 25 mm x 95 mm collection vial containing food.
2. Store vials containing flies horizontally to avoid trapping flies in any liquids that may accumulate in the bottom the vial.
3. Incubate flies for at least 21 hr at 22 °C at 45% humidity in an incubator for approximately for 15 hr. Set the incubator with a 12 hr light : dark cycle.

2. Climbing Assay

1. The following morning, transfer 20 flies from a single vial into a 250 ml glass graduated cylinder. Mark the position of the cylinder to keep it constant everyday. Use one glass cylinder per genotype to prevent cross contamination between the genotypes. Wash at the end of each experiment and rotate them between genotypes.
   1. Conduct the experiments in ambient light (or red light if there is a potential defect in vision) at temperature and humidity of 22 °C and 40% respectively. To avoid circadian rhythm confound, always perform experiments at the same time of day.
2. Seal the top of cylinder with a barrier film (wax film) to prevent the escape of any flies (Figure 2).
3. Set up the video camera on a tripod. Focus camera on the 190 ml line of the 250 ml graduated cylinder (17.5 cm).
4. Count the number of dead flies at the bottom of the cylinder and in the food vials. Record this number as the mortality.
5. Very lightly tap the cylinder against a closed cell foam pad repeatedly with enough force to displace the flies to the inner bottom surface. Tap 5 - 10 times while using the other hand to press record on the camera.
6. Press the “Record” button on the camera.
7. Start the video camera recording and tap the cylinder six times in a distinct non-rhythmic pattern.
8. Conduct each trial for 2 min from the time the flies are last tapped and record the number of flies crossing the height of 17.5 cm (190 ml) at each time point chosen (quantify every 10 sec). Note: The ml marking on the cylinder will vary from one cylinder model to another depending on diameter. To avoid error, measure the height on each cylinder used.
9. Once the trial has ended, dispose of flies in 95% ethanol.
10. Repeat steps 2.1 to 2.9 until all the replicates have been tested with fresh flies every time.
    Note: Although 5 replicates may be enough with a mutation having a strong effect on locomotion, 10 biological replicates of 20 flies (200 flies) is recommended to detect smaller differences.
11. Upon completion of the experiment, wash the cylinders in the lab dishwasher and dry O/N to be re-used.

3. Analysis

1. Analyze videos of each fly trial. Each 10 sec, record the total number of flies that pass the target line.
   1. If a fly climbs back down or falls, record that fly as -1 and count the next fly to cross the target line as the same number as the fly that climbed back down or fell. For example, if the 15^th fly falls below the target line, the next fly to cross the line (the 16^th fly) is considered the 15^th fly and not the 16^th.
2. Subtract the mortality from the total number of flies (20) to obtain the number of flies that remain in the trial. At each time point, obtain the fraction of flies above the target line.
3. Plot each percentage at each time point (see Figure 3).
4. Analyze the performance at the 120 sec data point and perform student t-test when 2 groups are present or ANOVA and a post-test for multiple comparisons (with Bonferroni modification for planned and Tukey for unplanned comparisons). The Kolmogorov-Smirnov tests¹⁷ is also performed to ascertain normality and equal variance but also to compare the distributions of the mutant group to the control.
5. To present the data over aging, plot the percentage of flies climbing at 120 sec with flies of different ages (2 days, 1 week, 2 weeks) to see if there is a progressive deficit (Figure 4).

Results

Climbing is a strong and reproducible behavior. Indeed, one day old wild-type flies reach the target distance climbing performance rapidly (25 - 30 sec). Mutant flies present a range of performance from mild (or delayed) to complete inability to climb to the target. We illustrate this here with two different mutant alleles. The first one is a severe allele of the gene spastin caused by a complete deletion of the spastin gene (spas^5.75) ¹⁸. In this line (spas^5.75 with TM6b) one day old fli...

Discussion

Drosophila has already proven to be an excellent model in Parkinson’s disease¹⁴ and other neurodegenerative conditions^1,2. In addition to the genetic tools available in Drosophila, its genome is highly conserved for genes involved in neurological disorders¹⁹. The advent of genome wide genetic screening methods (including whole exome sequencing) is likely to continue to provide a larger list of candidate genes associated with human movement disorders. The developmen...

Materials

Name	Company	Catalog Number	Comments
Drosophila stocks			The stocks are selected depending on the experiments. The temperature and humidity in the room and in the incubator must be controled and consistent to avoid flies being too staticky or too wet.
Video camera			Any digital camcorder will do. Make sure they can focus on close object.
Graduated cylinder	Kimble	20028W	Different models of graduated cylinder may have different diameter. It is therefore imporant to measure the height.
Computer			Any model will do. We used the computer to monitor the climbing of the flies and record the number of flies at each time point.