Name: an assay for measuring the effects of ethanol on the locomotion speed of caenorhabditis elegans
Uploaded: 2015-04-09T13:00:00.000+00:00
Duration: 10 min 35 s
Description: Watch this Scientific Journal Video about An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans at JoVE.com

These studies were supported by grants from the National Institutes of Health, National Institute for Alcoholism and Alcohol Abuse: R01AA016837 (JCB) and P20AA017828 (AGD and JCB).

1. Steps to Perform on the Day before the Assay
<ol>
	<li>Pick L4 stage worms to fresh nematode growth medium (NGM) plates seeded with a lawn of OP50 E. coli, and culture them at 20 &#730;C O/N. Each assay condition requires 10 worms; pick an excess of worms to allow for O/N loss of worms.
	<ol>
		<li>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.</li>
	</ol>
	</li>
</ol>
2. Steps to Perform on the Day of the Assay
<ol>
	<li>Preparation for the assay:
	<ol>
		<li>Perform assays on standard unseeded 60 x 15 mm NGM plates. Dry all of the plates to be used at 37 &#730;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.</li>
		<li>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&#8211;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.</li>
		<li>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 &#8216;skipping&#8217;.
		<ol>
			<li>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.</li>
			<li>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.</li>
			<li>For each assay plate prepare an accompanying &#8220;acclimation&#8221; plate, which should be dried and unseeded and will receive no ethanol. Place four copper rings on these plates.</li>
		</ol>
		</li>
		<li>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.</li>
		<li>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.</li>
		<li>When 1.5 hr has elapsed, begin the acclimation step of the assay, step 2.2.</li>
	</ol>
	</li>
	<li>Perform locomotion assay: Assay plates
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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 &#60; 1.5 min, but any interval up to 2 min&#160;is acceptable. The standard assay has movies recording at 10-12 min&#160;and 30-32 min&#160;of exposure for both 0 mM and 400 mM ethanol.</li>
		<li>Initiate the acclimation plate for the 0 mM exposure and the acclimation plate for the 400 mM exposure approximately 2 min&#160;and 30 sec&#160;apart to allow for saving of the first movie file before the second movie must begin.</li>
		<li>After the 30-min&#160;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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Perform Locomotion Assay: Filming
	<ol>
		<li>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 &#181;m pixel CCD.</li>
		<li>Use even illumination across the field of view, which aids in object recognition at the intensity threshold step (see below). A 3&#8221;x3&#8221; backlight works well.</li>
		<li>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.</li>
		<li>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&#160;(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.</li>
		<li>Record the movies. Begin recording a 120-frame movie of the first plate (0 mM ethanol) 10 min&#160;after the last worms were placed on that plate. Save the movie file.</li>
		<li>Record the second plate (400 mM ethanol). Repeat this process for both plates beginning 30 min&#160;after the last worms were placed on the first plate (0 mM ethanol) to capture the 30&#8211;32 min&#160;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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Analysis of movies using ImagePro software.
	<ol>
		<li>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.</li>
		<li>Analyze movies in 2-min&#160;(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&#62;Process&#62;Filters&#62;Enhancement&#62;Flatten: Parameters: Background = Dark; Feature Width = 20 Pix.</li>
		<li>Re-save the movies after the filtering step but always retain the original movie in its unfiltered form.
		<ol>
			<li>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&#62;Measure&#62;Track Objects&#8230; 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.</li>
		</ol>
		</li>
		<li>Under the Auto Tracking tab, use the following parameters: Track Parameters: Velocity limit (search radius) = 400 &#181;m/frame, Acceleration limit = Auto, Minimum total track length = 400 &#181;m, Predominant motion type = chaotic; Objects in tracks parameters: Allow partial tracks = yes, Min length = 21 frames, Tracking prediction depth = 1 frame.</li>
		<li>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.
		<ol>
			<li>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&#8211;1,500 on a scale of 0&#8211;4,095 is a good starting point for finer tuning.</li>
			<li>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&#62;Select Measurements menu item on the Count/Size options dialog box. (Area range: 28,000&#8211;120,000 &#181;m2 and Perimeter range: 600&#8211;2,500 &#181;m).</li>
			<li>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.</li>
		</ol>
		</li>
		<li>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.</li>
		<li>Use the software to calculate the velocity of each worm between each frame (distance traveled for the centroid of an object per 1 sec&#160;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.</li>
		<li>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.</li>
		<li>Calculate relative speed of locomotion through:</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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 ethanol8,10,20,24,25. This method allows the differentiation and simultaneous examination of two different ethanol response behavior phenotypes, initial sensitivity and the development of ac...

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 environment1,2. A strong physiological predictor of abuse liability is the initial level of response (LR) to alcohol (ethanol) that is exhibited by na&#239;ve drinkers3-5. 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 behavior7-9. 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 mammals10-16, and have been implicated in abuse liability in humans17-19. 
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 swimming11,12,14,20,21) or in the composition of the solutions in which the assays are performed (nematode growth medium versus Dent&#8217;s saline20,22). 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 swimming9,23, 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 ethanol8,10,20,24,25. The mammalian LR phenotype is a composite of at least two components, initial sensitivity to ethanol and acute functional tolerance to ethanol26,27. 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.

<table><tbody><tr><td>C. elegans strains</td><td>Caenorhabditis Genetics Center</td><td></td><td></td></tr><tr><td>60 x 15 mm Petri plates, triple vented</td><td>Greiner Bio-One</td><td>628161</td><td>Other plate brands will suffice.</td></tr><tr><td>NGM agar</td><td>Various</td><td></td><td>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) MgSO&lt;sub&gt;4&lt;/sub&gt;, 1 ml (1 M) CaCl&lt;sub&gt;2&lt;/sub&gt;, 25 ml (1 M) KPO&lt;sub&gt;4&lt;/sub&gt;, pH=6, 975 ml H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Forceps</td><td>Various</td><td></td><td>&lt;I&gt;e.g.&lt;/I&gt;, Fisher Scientific #10300</td></tr><tr><td>37 &amp;deg;C Incubator</td><td>Various</td><td></td><td>For drying agar</td></tr><tr><td>Digital balance</td><td>Various</td><td></td><td>For determining plate weights and agar volume</td></tr><tr><td>Copper rings</td><td>Plumbmaster</td><td>STK#35583 (48 cap thread gasket)</td><td>1.6 cm inner diameter, 1.8 cm outer diameter copper rings</td></tr><tr><td>100% ethanol</td><td>Various</td><td></td><td></td></tr><tr><td>Parafilm M</td><td>Bemis</td><td>PM996</td><td></td></tr><tr><td>CCD camera</td><td>QImaging</td><td>RET-4000R-F-M-12</td><td>This camera has a large field of view.</td></tr><tr><td>Stereomicroscope with C-mount and 0.5X objective</td><td>Leica</td><td>MZ6</td><td>Discontinued model, M60 is current equivalent.</td></tr><tr><td>Light source</td><td>Schott</td><td>A08923</td><td>3&amp;rdquo;x3&amp;rdquo;&amp;nbsp; backlight for even illumination across the field of view</td></tr><tr><td>Imaging and tracking software</td><td>Media Cybernetics</td><td>ImagePro-Plus v6.0-6.3</td><td>Newer versions of the software have tracking functions.</td></tr></tbody></table>

an assay for measuring the effects of ethanol on the locomotion speed of caenorhabditis elegans

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.&#160;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.&#160;elegans.

Representative data (Figure 1) from several different genotypes and their paired controls are presented8,24; 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&#160;are considered to be ethanol resistant (Figure 1F, G), w...

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

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.

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

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the ...

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Research

JoVE Journal

Neuroscience

8.6K Views.  Virginia Commonwealth University. 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. 

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

A Simple Way to Measure Ethanol Sensitivity in Flies

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain is correlated with that strain s ethanol preference, and therefore sedation is a highly relevant measure to study the genetics of alcohol responses and drinking. We demonstrate a simple way to expose flies to ethanol and measure its intoxicating effects. The assay we describe can determine acute sensitivity, as well as ethanol tolerance induced by repeat exposure. It does not require a technically involved setup, and can therefore be applied in any laboratory with basic fly culture tools.

A simple assay to measure the sedating effects of ethanol on Drosophila flies, based on the loss of righting reflex, is described.

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain ...

Measuring Caenorhabditis elegans Sensitivity to the Acetylcholine Receptor Agonist Levamisole

At the neuromuscular junction (NMJ), the binding of the excitatory neurotransmitter acetylcholine (ACh) to postsynaptic receptors leads to muscle contraction. As in vertebrate skeletal muscle, cholinergic signaling in the body wall muscles of the model organism Caenorhabditis elegans is required for locomotion. Exposure to levamisole, a pharmacological agonist of one class of ACh receptors on the body wall muscles, causes time-dependent paralysis of wild-type animals. Altered sensitivity to levamisole suggests defects in signaling at the NMJ or muscle function. Here, a protocol for a liquid levamisole assay performed on C. elegans grown in 24-well plates is presented. Vigorous swimming of the animals in liquid allows for the assessment and quantitation of levamisole-induced paralysis in hundreds of worms over a one-hour time period without requiring physical manipulation. This procedure can be used with both wild-type and mutants that have altered sensitivity to levamisole to demonstrate the functional consequences of altered signaling at the NMJ.

Measuring Caenorhabditis elegans Sensitivity to the Acetylcholine Receptor Agonist Levamisole

The present protocol describes an assay to determine response to levamisole, a pharmacological agonist of one class of Caenorhabditis elegans acetylcholine receptors. In this liquid levamisole swim assay, researchers visually observe and quantitate the time-dependent paralysis of animals cultivated in 24-well plates.

At the neuromuscular junction (NMJ), the binding of the excitatory neurotransmitter acetylcholine (ACh) to postsynaptic receptors leads to muscle ...

Genetics

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Caenorhabditis elegans is a simple genetic organism amenable to large-scale forward and reverse genetic screens and chemical genetic screens. The C. elegans genome includes potential antipsychotic drug (APD) targets conserved in humans, including genes encoding proteins required for neurotransmitter synthesis and for synaptic structure and function. APD exposure produces developmental delay and/or lethality in nematodes in a concentration-dependent manner. These phenotypes are caused, in part, by APD-induced inhibition of pharyngeal pumping1,2. Thus, the developmental phenotype has a neuromuscular basis, making it useful for pharmacogenetic studies of neuroleptics. Here we demonstrate detailed procedures for testing APD effects on nematode development and pharyngeal pumping. For the developmental assay, synchronized embryos are placed on nematode growth medium (NGM) plates containing APDs, and the stages of developing animals are then scored daily. For the pharyngeal pumping rate assay, staged young adult animals are tested on NGM plates containing APDs. The number of pharyngeal pumps per unit time is recorded, and the pumping rate is calculated. These assays can be used for studying many other types of small molecules or even large molecules.

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Approaches for testing the effects of antipsychotic drugs (APDs) in Caenorhabditis elegans are demonstrated. Assays are described for testing drug effects on development and viability and on pharyngeal pumping rate. These methods are also applicable for pharmacogenetic experiments with drug classes other than APDs.

Caenorhabditis elegans is a simple genetic organism amenable to  large-scale forward and reverse genetic screens and chemical genetic screens. The C.  ...

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

Drosophila melanogaster provides an excellent model to study the genetic underpinnings of alcohol sensitivity. In contrast to studies in human populations, the Drosophila model allows strict control over genetic background, and virtually unlimited numbers of individuals of the same genotype can be reared rapidly under well-controlled environmental conditions without regulatory restrictions and at relatively low cost. Flies exposed to ethanol undergo physiological and behavioral changes that resemble human alcohol intoxication, including loss of postural control, sedation, and development of tolerance. Here, we describe a simple, low-cost, high-throughput assay for assessing alcohol sedation sensitivity in large numbers of single flies. The assay is based on video recording of single flies introduced without anesthesia in 24-well cell culture plates in a set-up that enables synchronous initiation of alcohol exposure. The system enables a single person to collect individual ethanol sedation data on as many as 2,000 flies within an 8 h work period. The assay can, in principle, be extended to assess the effects of exposure to any volatile substance and applied to measure effects of acute toxicity of volatiles on other insects, including other fly species.

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

Current methods to measure alcohol sensitivity in Drosophila are designed to test groups of flies. We present a simple, low-cost, high-throughput assay for assessing alcohol sedation sensitivity in large numbers of single flies. The method does not require specialized tools and can be performed in any laboratory using common materials.

Drosophila melanogaster provides an excellent model to study the genetic underpinnings of alcohol sensitivity. In contrast to studies in human ...

Behavior

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease

Caenorhabditis elegans is widely recognized for its central utility as a translational animal model to efficiently interrogate mechanisms and therapies of diverse human diseases. Worms are particularly well-suited for high-throughput genetic and drug screens to gain deeper insight into therapeutic targets and therapies by exploiting their fast development cycle, large brood size, short lifespan, microscopic transparency, low maintenance costs, robust suite of genomic tools, mutant repositories, and experimental methodologies to interrogate both in vivo and ex vivo physiology. Worm locomotor activity represents a particularly relevant phenotype that is frequently impaired in mitochondrial disease, which is highly heterogeneous in causes and manifestations but collectively shares an impaired capacity to produce cellular energy. While a suite of different methodologies may be used to interrogate worm behavior, these vary greatly in experimental costs, complexity, and utility for genomic or drug high-throughput screens. Here, the relative throughput, advantages, and limitations of 16 different activity analysis methodologies were compared that quantify nematode locomotion, thrashing, pharyngeal pumping, and/or chemotaxis in single worms or worm populations of C. elegans at different stages, ages, and experimental durations. Detailed protocols were demonstrated for two semi-automated methods to quantify nematode locomotor activity that represent novel applications of available software tools, namely, ZebraLab (a medium-throughput approach) and WormScan (a high-throughput approach). Data from applying these methods demonstrated similar degrees of reduced animal activity occurred at the L4 larval stage, and progressed in day 1 adults, in mitochondrial complex I disease (gas-1(fc21)) mutant worms relative to wild-type (N2 Bristol) C. elegans. This data validates the utility for these novel applications of using the ZebraLab or WormScan software tools to quantify worm locomotor activity efficiently and objectively, with variable capacity to support high-throughput drug screening on worm behavior in preclinical animal models of mitochondrial disease.

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease

This study presents protocols for two semi-automated locomotor activity analysis approaches in C. elegans complex I disease gas-1(fc21) worms, namely, ZebraLab (a medium-throughput assay) and WormScan (a high-throughput assay) and provide comparative analysis among a wide array of research methods to quantify nematode behavior and integrated neuromuscular function.

Caenorhabditis elegans is widely recognized for its central utility as a translational animal model to efficiently interrogate mechanisms and therapies of ...

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in their etiology. The fruit fly, Drosophila melanogaster, is a powerful model for exploring molecular-genetic mechanisms underlying alcohol-related behaviors and therefore holds great promise for identifying and understanding the function of genes that influence AUD. The use of the Drosophila model for these types of studies depends on the availability of assays that reliably measure behavioral responses to ethanol. This report describes an assay suitable for assessing ethanol sensitivity and rapid tolerance in flies. Ethanol sensitivity measured in this assay is influenced by the volume and concentration of ethanol used, a variety of previously reported genetic manipulations, and also the length of time the flies are housed without food immediately prior to testing. In contrast, ethanol sensitivity measured in this assay is not affected by the vigor of fly handling, sex of the flies, and supplementation of growth medium with antibiotics or live yeast. Three different methods for quantitating ethanol sensitivity are described, all leading to essentially indistinguishable ethanol sensitivity results. The scalable nature of this assay, combined with its overall simplicity to set-up and relatively low expense, make it suitable for small and large scale genetic analysis of ethanol sensitivity and rapid tolerance in Drosophila.

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Straightforward assays for measuring ethanol sensitivity and rapid tolerance in Drosophila facilitate the use of this model organism for investigating these important ethanol-related behaviors. Here, a relatively simple, scalable assay for measuring ethanol sensitivity and rapid tolerance in flies is described.

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

Biology

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

Touch Habituation Assay: A Method to Study Non-Associative Learning in C. elegans

Source: Mersha, M. D., et al. <a href="https://www.jove.com/video/56771/long-term-behavioral-reproductive-consequences-embryonic-exposure-to">Long-term Behavioral and Reproductive Consequences of Embryonic Exposure to Low-dose Toxicants.</a> J. Vis. Exp. (2018).
This video introduces the touch habituation assay, a C. elegans behavioral method of assessing mechanosensation and quantifying time to habituation.

Test habituacji dotyku: metoda badania uczenia się bez skojarzeń u C. elegans

Source: Mersha, M. D., et al. Long-term Behavioral and Reproductive Consequences of Embryonic Exposure to Low-dose Toxicants. J. Vis. Exp. (2018).
This ...

JoVE Encyclopedia of Experiments

Caenorhabditis elegans (worm)

Electrotaxis Assay: A Method to Observe Locomotion in C. elegans

Source: Tong, J,&#160;et al. <a href="https://www.jove.com/video/50226/microfluidic-based-electrotaxis-for-on-demand-quantitative-analysis">Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of&#160;Caenorhabditis elegans&#39; Locomotion.</a>&#160;J. Vis. Exp.&#160;(2013).
Researchers can trigger on-demand movement in C. elegans by applying an electric field that worms sense and respond to.&#160; This video introduces electrotactic behavior and shows a sample protocol that is done in a microfluidic device.

Test elektrotaksji: metoda obserwacji lokomocji u C. elegans

Source: Tong, J,&#160;et al. Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of&#160;Caenorhabditis elegans&#39; Locomotion.&#160;J. ...

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

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A Simple Way to Measure Ethanol Sensitivity in Flies

Measuring Caenorhabditis elegans Sensitivity to the Acetylcholine Receptor Agonist Levamisole

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

C. elegans Chemotaxis Assay

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

Test habituacji dotyku: metoda badania uczenia się bez skojarzeń u C. elegans

Test elektrotaksji: metoda obserwacji lokomocji u C. elegans