An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

Summary

C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay.

Abstract

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the development of more effective treatments is the relative lack of understanding of the molecular underpinnings of the effects of ethanol on behavior. The nematode, Caenorhabditis elegans (C. elegans), provides a useful model in which to generate and test hypotheses about the molecular effects of ethanol. Here, we describe an assay that has been developed and used to examine the roles of particular genes and environmental factors in behavioral responses to ethanol, in which locomotion is the behavioral output. Ethanol dose-dependently causes an acute depression of crawling on an agar surface. The effects are dynamic; animals exposed to a high concentration demonstrate an initial strong depression of crawling, referred to here as initial sensitivity, and then partially recover locomotion speed despite the continued presence of the drug. This ethanol-induced behavioral plasticity is referred to here as the development of acute functional tolerance. This assay has been used to demonstrate that these two phenotypes are distinct and genetically separable. The straightforward locomotion assay described here is suitable for examining the effects of both genetic and environmental manipulations on these acute behavioral responses to ethanol in C. elegans.

Introduction

Alcohol use disorders (AUD) are widespread and produce serious health, social, and economic problems. In humans, the susceptibility to developing an AUD is heavily influenced by both genetics and the environment^1,2. A strong physiological predictor of abuse liability is the initial level of response (LR) to alcohol (ethanol) that is exhibited by naïve drinkers^3-5. This LR phenotype is influenced by genetics and non-genetic components⁶. Determining the molecular mechanisms that influence the LR to ethanol is an important goal of the study of ethanol response behaviors.

The nematode, Caenorhabditis elegans, has been increasingly used as a model for studying the effects of ethanol on behavior^7-9. There is strong molecular conservation in the machinery of nervous system function between worms and mammals, and several genes that have been shown to influence the LR to ethanol in worms have been shown to influence LR to ethanol in mammals^10-16, and have been implicated in abuse liability in humans^17-19.

Ethanol intoxicates worms, which is reflected in a decrease in their locomotion speed. Several different laboratories have developed behavioral assays that differ in several ways, for example, the locomotion behavior that they study (crawling versus swimming^{11,12,14,20,21}) or in the composition of the solutions in which the assays are performed (nematode growth medium versus Dent’s saline^20,22). Interestingly, these diverse assays have yielded somewhat different dose response profiles for the effects of ethanol. These results have pointed to important differences in the underlying behaviors of crawling and swimming^9,23, as well as a role for the environmental variable osmolarity in ethanol responses²⁰, and have highlighted the importance of describing experimental detail of the various assays.

An assay to measure the acute effects of ethanol on crawling behavior is presented here. This assay has been used extensively to study the genetic and environmental influences on the LR to ethanol^{8,10,20,24,25}. The mammalian LR phenotype is a composite of at least two components, initial sensitivity to ethanol and acute functional tolerance to ethanol^26,27. In worms, the LR phenotype has been shown to be separable into these two components through the use of this behavioral assay. The influences of genetic and environmental manipulations on both phenotypes can be examined using this single assay. Importantly, these two phenotypes are genetically separable.

Protocol

1. Steps to Perform on the Day before the Assay

1. Pick L4 stage worms to fresh nematode growth medium (NGM) plates seeded with a lawn of OP50 E. coli, and culture them at 20 ˚C O/N. Each assay condition requires 10 worms; pick an excess of worms to allow for O/N loss of worms.
   1. Only assay animals that are first-day adults; many mutants grow at a slower speed than wild type. Adjust the timing of picking for strains that have developmental delays so that all animals tested are first-day adults.

2. Steps to Perform on the Day of the Assay

1. Preparation for the assay:
   1. Perform assays on standard unseeded 60 x 15 mm NGM plates. Dry all of the plates to be used at 37 ˚C for 2 hr, with lids off. For each experimental trial, use four dried NGM plates; these will be the 0 mM and 400 mM ethanol assay plates and their accompanying acclimation plates.
      NOTE: The use of NGM plates is important here; differences in the composition of the plates, in particular their osmolarity, can strongly affect the dose response of ethanol on behavior, this is due, at least in part, to changes in the amount of ethanol accumulated by the animals²⁰. NGM agar is 160 mOsm.
   2. After drying, weigh each of the unseeded NGM plates to be used in the assay and note the weight. Determine the volume of media in the plates based on the weight of an empty plate. To approximate the conversion of weight of the agar to volume, assume that the media weighs the same as an equal volume of water.
      NOTE: Our most consistent results have been found with NGM media that has dehydrated sufficiently that an original 10 ml volume has a post-drying volume between 8.3–8.9 ml. An alternative to the 2 hr dry time is to dry until the plates reach this volume range to account for differences in incubators.
   3. Melt 4 copper rings (inner diameter of 1.6 cm) into the surface of each of the plates to act as corrals for the different genotypes or treatment groups of worms.Grasp each ring with forceps, and heat in a flame (a strong flame from a Bunsen burner works well) for approximately 3 sec. Immediately place the ring on a plate while still holding the ring with the forceps to prevent it from ‘skipping’.
      1. Ensure that the ring rests flat against the surface of the agar by pressing down gently with the forceps at several points on the ring. When placing the ring, be careful to leave room for an additional three rings.
      2. Melt the three additional copper rings into the surface of the plate, taking care to place them as close together as possible so that all four will be in the field of view of the camera.
         NOTE: Making a good seal with the agar is essential to keep the worms in the rings during the assay. Rings that skip around on the plate are unlikely to make the correct seal with the agar and may scar the agar, which allows worms to burrow and interferes with visualizing the worms.
      3. For each assay plate prepare an accompanying “acclimation” plate, which should be dried and unseeded and will receive no ethanol. Place four copper rings on these plates.
   4. Label the bottom of the plates next to each ring with the worm strain to be used in the ring, taking care to not write in the field of view of the ring itself. Match the labels on the assay plate with its accompanying acclimation plate.
   5. Calculate the amount of 100% ethanol to add to each assay plate so that the final concentration is 0 mM or 400 mM ethanol (weight to volume). For each experiment (n = 1), there will be one 0 mM and one 400 mM ethanol plate; acclimation plates do not receive ethanol. Add the ethanol, pipetting it around the surface of the plate. Use laboratory film to seal the plate and allow it to equilibrate on the bench top for 2 hr.
   6. When 1.5 hr has elapsed, begin the acclimation step of the assay, step 2.2.
2. Perform locomotion assay: Assay plates
   1. Carefully transfer 10 worms of each experimental group to the center of a copper ring on the acclimation plate. Remove any food that is visible on the agar at this point by scraping gently with the worm pick. Vary the order in which the experimental groups are put on the plates across experimental trials so that the same strains are not put on the plates in the same order across trials.
      NOTE: The goal is to transfer the animals to the plate with minimal quantities of food, because if food on the acclimation plate is transferred to the assay plate the worms will aggregate around the food and the effect of the drug on locomotion will be obscured.
   2. Be careful not to break the surface of the agar with the pick, as this will allow the worms to burrow and will disrupt the assay. Incubate the worms at RT for 30 min.
   3. Ensure that there is an appropriate interval between the initiations of each plate. An experienced experimenter can move 40 animals, 10/ring for 4 rings in < 1.5 min, but any interval up to 2 min is acceptable. The standard assay has movies recording at 10-12 min and 30-32 min of exposure for both 0 mM and 400 mM ethanol.
   4. Initiate the acclimation plate for the 0 mM exposure and the acclimation plate for the 400 mM exposure approximately 2 min and 30 sec apart to allow for saving of the first movie file before the second movie must begin.
   5. After the 30-min acclimation period, transfer the worms from the acclimation plates to the assay plates. Transfer the worms to the assay plates (0 mM or 400 mM ethanol) in the same order that they were added to the acclimation plate, keeping track of the timing between the completions of each plate. Seal the plate with laboratory film to minimize loss of ethanol to vaporization.
   6. Use a scooping motion with a thin-edged flattened worm pick to collect worms on top of the flattened pick. Perform the transfer of worms from the unseeded acclimation plate to the assay plate without the use of bacteria, which is commonly used in transferring worms as it helps to glue the worm to the pick.
      NOTE: The speed at which animals are transferred at this step is very important because the first worms on the plate will be exposed to ethanol longer than the last worms added to the plate, and earlier time points may show some time-dependent effects. This is the major reason for rotating which strains are placed on a plate first across experimental replicates.
3. Perform Locomotion Assay: Filming
   1. Use a microscope/camera combination that allows for simultaneous imaging of all four rings in the field of view (approximately 42x42 mm² square), such as a 0.5x microscope objective, 0.8x magnification and a camera with a 2048x2048 7.4 µm pixel CCD.
   2. Use even illumination across the field of view, which aids in object recognition at the intensity threshold step (see below). A 3”x3” backlight works well.
   3. Image the worms on a plate positioned media-side up (lid down), which generates contrast that is lost when using the backlight compared with a traditional microscope transmitted light source.
   4. Prepare the image analysis software to capture time-lapse movies of the moving worms. Set the software to capture 12-bit gray-scale images every 1 sec, as a 2-min (120 frame) movie. To reduce the size of the output file, while still retaining sufficient image resolution, use a 2x2 binning mode to capture 1024x1024 pixel images.
   5. Record the movies. Begin recording a 120-frame movie of the first plate (0 mM ethanol) 10 min after the last worms were placed on that plate. Save the movie file.
   6. Record the second plate (400 mM ethanol). Repeat this process for both plates beginning 30 min after the last worms were placed on the first plate (0 mM ethanol) to capture the 30–32 min time points for each exposure.
      NOTE: Allow sufficient time to save the image files after each capture session before beginning recording of the next plate to be analyzed.
   7. For future-proofing of archived movies and to allow these movies to be opened and analyzed by other public domain open source imaging software programs, such as ImageJ²⁸, convert a copy of each movie to 8-bit 256 gray scale TIFF files.
      NOTE: The image analysis software described here uses a proprietary file format.
4. Analysis of movies using ImagePro software.
   1. Once captured, analyze the movies using Image-Pro Plus software or its equivalent.
      NOTE: A variety of other object tracking software is available that has been used to successfully track multiple C. elegans at one time²⁹ and such software could reasonably substitute for that described here as the object identification steps are based on similar principles. The method described relates to Image-Pro Plus software versions 6.0-6.3 and 7.0, newer versions of Image-Pro Plus software may have minor differences.
   2. Analyze movies in 2-min (120 frame) segments. First, apply a filter to the images to flatten the background and enhance the contrast of the worm objects. Select the filter as follows: Menu>Process>Filters>Enhancement>Flatten: Parameters: Background = Dark; Feature Width = 20 Pix.
   3. Re-save the movies after the filtering step but always retain the original movie in its unfiltered form.
      1. Analyze the locomotion of animals in each ring separately with a circular region of interest (ROI) that is placed and sized to overlap with the copper ring. Identify and track the worms with the Menu>Measure>Track Objects… command; this brings up the Tracking Data Table window. The Tracking Options button allows specific tracks to be excluded and to limit any experimental artifacts.
   4. Under the Auto Tracking tab, use the following parameters: Track Parameters: Velocity limit (search radius) = 400 µm/frame, Acceleration limit = Auto, Minimum total track length = 400 µm, Predominant motion type = chaotic; Objects in tracks parameters: Allow partial tracks = yes, Min length = 21 frames, Tracking prediction depth = 1 frame.
   5. To initiate the tracking process, click the Find All Tracks Automatically function button to bring up the Count/Size options dialog box and the Tracking dialog box. Select the Manual option for the Intensity Range Selection in the Count/Size dialog box, which provides the important threshold step that uses the gray-scale intensity of the worm pixels to highlight a worm object for analysis and to ignore the lighter background pixels.
      1. Adjust the intensity threshold sliders (one to set the upper limit, one to set the lower limit) to create an inclusive range that highlights all dark objects. A range of 0–1,500 on a scale of 0–4,095 is a good starting point for finer tuning.
      2. Apply a size filter to exclude objects that are larger and smaller than a single worm object, such as the copper ring and any debris on the plates. Set two size parameters to do this that cover sizes for individual worms in a variety of postures with the Measure>Select Measurements menu item on the Count/Size options dialog box. (Area range: 28,000–120,000 µm² and Perimeter range: 600–2,500 µm).
      3. If a mutant strain is significantly smaller or larger than other animals on the plate to be analyzed, broaden the range of the filter settings for object recognition to accommodate the different sizes of the strains first before tracking animals so that settings do not need to be changed mid-analysis.
   6. Complete the tracking process by clicking Continue in the Tracking dialog box. Visually compare the output tracks with the progress of each worm in the movie to ensure that every worm is represented unless there is a valid reason to exclude it based on the automatic filter settings. Manually delete tracks that were produced by the presence of confirmed non-worm objects that met the size filter criteria.
   7. Use the software to calculate the velocity of each worm between each frame (distance traveled for the centroid of an object per 1 sec between frames) and display the average velocity for each track and the average velocity across all tracks for the population of worms in the copper ring. Consider this final average to be n = 1 in terms of experimental trials. Export the data to a spreadsheet program for statistical analyses and data archiving.
   8. Once the data has been recorded for the first ring on the plate, move the ROI to the next ring and repeat the tracking process, without changing any of the parameters.
   9. Calculate relative speed of locomotion through:
   10. Relative speed (%) = treated (400 mM) speed/ untreated (0 mM) speed x 100.
       NOTE: Different genetic manipulations often alter the basal locomotion rate of animals. In order to determine the effect of ethanol on the locomotion speed of animals and to be able to compare these effects across different genotypes or conditions, calculate a relative speed.

Results

Representative data (Figure 1) from several different genotypes and their paired controls are presented^8,24; data were specifically chosen that highlight differences in the assayed animals. The degree of effect at 10 min of exposure is considered the initial sensitivity of a strain, which is shown on the left axes in Figure 1B-G. Mutant strains with a relative speed larger than the control at 10 min are considered to be ethanol resistant (Figure 1F, G), while ...

Discussion

The simple neurobiology and genetic tools available in C. elegans make the worm an excellent model in which to study the molecular bases of the effects of ethanol on behavior. Here, we describe an assay that has been used to identify several molecular and environmental mediators of the acute behavioral response to ethanol^{8,10,20,24,25}. This method allows the differentiation and simultaneous examination of two different ethanol response behavior phenotypes, initial sensitivity and the development of ac...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
C. elegans strains	Caenorhabditis Genetics Center
60 x 15 mm Petri plates, triple vented	Greiner Bio-One	628161	Other plate brands will suffice.
NGM agar	Various		NaCl (3 g/L), agar (17 g/L), peptone (2.5 g/L), 1 ml cholesterol (5 mg/ml in ethanol), 1 ml (1 M) MgSO4, 1 ml (1 M) CaCl2, 25 ml (1 M) KPO4, pH=6, 975 ml H2O
Forceps	Various		e.g., Fisher Scientific #10300
37 °C Incubator	Various		For drying agar
Digital balance	Various		For determining plate weights and agar volume
Copper rings	Plumbmaster	STK#35583 (48 cap thread gasket)	1.6 cm inner diameter, 1.8 cm outer diameter copper rings
100% ethanol	Various
Parafilm M	Bemis	PM996
CCD camera	QImaging	RET-4000R-F-M-12	This camera has a large field of view.
Stereomicroscope with C-mount and 0.5X objective	Leica	MZ6	Discontinued model, M60 is current equivalent.
Light source	Schott	A08923	3”x3”  backlight for even illumination across the field of view
Imaging and tracking software	Media Cybernetics	ImagePro-Plus v6.0-6.3	Newer versions of the software have tracking functions.