Name: Measuring Associative Learning in Chemotaxis of the Nematode Caenorhabditis elegans (Video) | JoVE Journal
Uploaded: 2025-06-17T12:20:00.000+00:00
Duration: 9 min 53 s
Description: Learn classical conditioning protocols to measure associative learning and chemotaxis in Caenorhabditis elegans. This method reveals neural mechanisms underlying age-related cognitive decline.

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5 minutes

We dedicate this paper to the memory of Johannes Berthold. Some strains were provided by the Caenorhabditis Genetics Center (CGC), which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440). E.I.B. gratefully acknowledges financial support by Studienstiftung des deutschen Volkes.

1. Preparation of growth plates (GP) to synchronize C. elegans prior to associative learning test, and conditioning plates (CP) to induce [NaCl]-food association on test day
NOTE: Petri dishes for growth and conditioning are identical to the standard nematode growth media (NGM) plates12, except that NaCl concentration was varied in the 0 mM NaCl CP and 100 mM NaCl CP plates.
<ol>
	<li>Prepare four clean 500 mL glass bottles and add the following:
	<ol>
		<li>Add 1.25 g of peptone, 8.5 g of agar, 487.5 mL of distilled water, and a magnetic stirrer to one bottle (for 0 mM NaCl CP);</li>
		<li>Add 1.5 g of NaCl, 1.25 g of peptone, 8.5 g of agar, 487.5 mL of distilled water, and a magnetic stirrer to each of two bottles (for 50 mM NaCl CP and GP, respectively);</li>
		<li>Add 3 g of NaCl, 1.25 g of peptone, 8.5 g of agar, 487.5 mL of distilled water, and a magnetic stirrer to one bottle (for 100 mM NaCl CP).</li>
	</ol>
	</li>
	<li>Autoclave the four bottles for 15 min at 121 °C, then let the bottles cool down for 30 min at 55 °C in a water bath.</li>
	<li>Place the bottles (one at a time) on a heated magnetic stirrer. Add to each bottle 12.5 mL of 1 M KPO4 (pH = 6.0 at room temperature [RT]). Mix thoroughly for 2 min, then add 0.5 mL of 1 M CaCl2, 0.5 mL of 5 mg/mL cholesterol in ethanol, and 0.5 mL of 1 M MgSO4 (all solutions must be sterile). Mix by magnetic stirring. 
	NOTE: Mixing thoroughly after adding KPO4 prevents the precipitation of calcium phosphate upon the addition of CaCl2.</li>
	<li>For each type of plate (0 mM CP, 50 mM CP, 100 mM CP, GP), aliquot 8 mL of the respective medium to each 55 mm x 10 mm Petri dish under near-sterile conditions (in a flow hood or next to Bunsen burner). 
	NOTE: Approximately 62 NGM plates of each type can be made from this step.</li>
	<li>Cool down the plates by leaving them at RT for approximately 3 h, then store them at 4 °C for up to 2 weeks until use.</li>
	<li>Add 200 µL of E. coli OP50 suspension to each NGM GP and allow to dry overnight at 20 °C in an incubator. 
	NOTE: The plates can be stored at RT for up to 1 week before being used.</li>
</ol>
2. Maintenance of C. elegans and preparation for associative learning test via timed egg-laying 13 (3 days before assay)
NOTE: One round of the chemotaxis assay in the suggested format requires at least 24 plates for growth (GP), 48 plates for the assay (Assay Plates, AP), and 8 plates per conditioning group (Conditioning Plates, CP). For a class split into eight student groups, this means each student group receives 3 GP, 6 CP, and 6 AP - i.e., 1 CP and 1 AP for each experimental condition.
<ol>
	<li>Maintain C. elegans N2 (var. Bristol) on NGM agar plates fed with live E. coli OP50 at 20 °C. Store the plates upside down throughout maintenance to minimize loss of moisture from the plates.</li>
	<li>Transfer 30 young (day 2) N2 adults onto each GP.</li>
	<li>Allow animals to lay eggs for approximately 8 h at 20 °C.</li>
	<li>Remove all adults from the plates and culture upside down at 20 °C for 3 days.</li>
</ol>
3. Preparation of assay (1 day before assay)
<ol>
	<li>Prepare seeded (+) and unseeded (-) CP.
	<ol>
		<li>Seed half of the batch of each of 0 mM NaCl CP, 50 mM NaCl CP, and 100 mM NaCl CP with 200 µL OP50 per plate (CP0+, CP50+, and CP100+, respectively). 
		NOTE: The addition of the OP50 suspension to CP0+ may result in carrying over a small amount of NaCl (&lt;2 mM NaCl), which is unlikely to substantially affect chemotaxis behavior. In situations where small concentrations of NaCl are relevant, this could be accommodated by adding 2 mM NaCl to the CP0- plates and the 'no salt' sector on the assay plates.</li>
		<li>Allow plates to dry overnight at 20 °C in an incubator alongside the unseeded half of the CP (CP0-, CP50-, and CP100-, respectively)</li>
	</ol>
	</li>
	<li>Prepare assay plates (AP).
	<ol>
		<li>Prepare three clean 1 L glass bottles and add the following:
		<ol>
			<li>Add 2.5 g of peptone, 17 g of agar, 975 mL of distilled water, and a magnetic stirrer to one bottle (for 0 mM NaCl)</li>
			<li>Add 3 g of NaCl, 2.5 g of peptone, 17 g of agar, 975 mL of distilled water, and a magnetic stirrer to one bottle (for 50 mM NaCl)</li>
			<li>Add 6 g of NaCl, 2.5 g of peptone, 17 g of agar, 975 mL of distilled water, and a magnetic stirrer to one bottle (for 100 mM NaCl)</li>
		</ol>
		</li>
		<li>Autoclave the three bottles for 15 min at 121 °C, then let the bottles cool down for 30 min at 55 °C in a water bath.</li>
		<li>Meanwhile, label the bottom of each section of the Y-Petri dish (a 90 mm x 10 mm Petri dish divided into three compartments by an internal wall) with the corresponding NaCl concentration of the medium to be poured into it. Draw a circle with a radius of 5 mm around the center of the plate, where the three walls meet (Figure 1). 
		NOTE: The area within the circle, along with the walls themselves, will represent the exclusion area for counting purposes: worms found within these areas during counting will not count towards any sector's total.</li>
		<li>Place the bottles (one at a time) on a heated magnetic stirrer. Add to each bottle 25 mL of 1 M KPO4 (pH = 6.0 at RT). Mix thoroughly for 2 min, then add 1 mL of 1 M CaCl2, 1 mL of 5 mg/mL cholesterol in ethanol, and 1 mL of 1 M MgSO4 (all solutions must be sterile). Mix by magnetic stirring.</li>
		<li>Aliquot the 0 mM medium into one compartment of a 90 mm x 10 mm Y-Petri dish until the medium reaches the top of the internal wall, then allow approximately 5 min to solidify.</li>
		<li>Aliquot the 100 mM medium into the second compartment of the Y-Petri dish until the medium touches the solidified 0 mM NaCl section adjacent to it. To avoid spillover into the adjacent empty section, fill the 100 mM section to the brim of the internal Y-wall, then use a sterile 10 µL pipette tip to break the surface tension of the medium along the Y-wall and connect it to the 0 mM NaCl section. Then, allow approximately 5 min to solidify.</li>
		<li>Aliquot the 50 mM medium into the remaining empty compartment of the Y-Petri dish until the medium touches the solidified 0 mM NaCl and 100 mM NaCl adjacent sections. Take care that the medium does not spill over into the adjacent sections. Then, allow approximately 5 min to solidify.</li>
		<li>Store the plates overnight at 20 °C in an incubator. 
		NOTE: Approximately 50 AP can be made from this method.</li>
	</ol>
	</li>
	<li>Prepare 1 L of sterile M9 buffer14.</li>
</ol>
4. C. elegans associative learning assay (here performed by 8 groups in parallel)
<ol>
	<li>Pipette 1 mL of sterile M9 buffer onto a GP and swirl the plate gently to dislodge the worms (mostly 1-day adults) from the OP50 and the agar, forming a suspension.</li>
	<li>Tilt the plate, collect the worm suspension in M9 buffer with a 1 mL plastic pipette tip, and transfer it to a 1.5 mL microcentrifuge tube. 
	NOTE: The top part of the pipette tip may be trimmed using a pair of scissors to increase the diameter of the opening; this reduces shear stress and physical damage as the worms pass through the tip and can be especially useful when working with 200 µL pipette tips. Before starting the experiments, it is important to ensure that the worms do not stick to the type of tube used; this can be done by observing the tube under a dissecting microscope after removing the worm suspension.</li>
	<li>Repeat the process for all GP, then let the worms settle to the bottom of the microcentrifuge tubes for approximately 60 s, forming a pellet.</li>
	<li>Remove the supernatant/excess M9 buffer using a 1 mL pipette, taking care not to disturb the pellet.</li>
	<li>Wash the pellet with 1 mL of sterile M9 buffer two further times to remove any residual OP50. Allow the pellet to settle for approximately 60 s between washes. Resuspend the pellet after washing it to 300 µL with M9 Buffer.</li>
	<li>Slowly transfer 50 µL of the worm suspension (approximately 200-300 animals) to each conditioning plate (CP0-, CP0+, CP50-, CP50+, CP100- and CP100+). Swirl the suspension each time before aliquoting to keep the worms evenly suspended. For plates seeded with OP50, place the worm droplet away from the bacterial food lawn. Allow the suspension to absorb into the agar.</li>
	<li>Store the plates upside down at 20 °C in an incubator for approximately 4 h, allowing the worms to roam. 
	NOTE: The conditioning step can be shortened to 2 h to accommodate undergraduate course schedules.</li>
	<li>Pipette 1 mL of sterile M9 buffer onto a CP and swirl the plate gently to dislodge the worms, forming a suspension.</li>
	<li>Tilt the plate, collect the M9-worm suspension with a 1 mL plastic pipette tip, and transfer it to a 1.5 mL microcentrifuge tube.</li>
	<li>Allow the worms to settle to the bottom of the microcentrifuge tube for approximately 60 s.</li>
	<li>Remove the supernatant/excess M9 buffer using a 1 mL pipette without disturbing the worm pellet.</li>
	<li>Wash the worms two further times with 1 mL of sterile M9 buffer. Allow the pellet to settle for approximately 60 s between washes.</li>
	<li>Fill the microcentrifuge tube with M9 buffer to the 1 mL mark and allow the worm pellet to settle for approximately 60 s.</li>
	<li>Remove 960 µL of the supernatant using a 1 mL pipette, taking care not to disturb the pellet.</li>
	<li>Swirl or shake the microcentrifuge tube gently to resuspend the worms, then use a 20 µL pipette to transfer 20 µL of the worm pellet (containing approximately 100-150 animals) to the center of an AP, where all segments of the Y-wall meet. Repeat this step for the other APs.</li>
	<li>Allow the worms to roam for 30 min at 20 °C, then count the number of worms present in each of the sections of the AP (0 mM NaCl, 50 mM NaCl, and 100 mM NaCl, respectively) under a stereo microscope. Exclude any worms present within 5 mm of the center of the plate in any section or on the border between sections (Figure 1).</li>
	<li>Allow the worms to roam for another 30 min at 20 °C, then count the number of worms in each section of the AP again. Exclude any worms present within 5 mm of the center of the plate in any section.</li>
	<li>Calculate the chemotaxis index (CI) per plate per time point using the formula15,16: 
	<img alt="Competition index formula; mathematical equation for worm distribution study analysis." src="/files/ftp_upload/66311/66311eq01.jpg" style="margin: auto;" data-altupdatedat="1785211204000" data-alt="Equation 1"></li>
</ol>

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</ol>

The authors declare that they have no conflict of interest.

In this article, we describe the evaluation of associative learning in the nematode C. elegans using chemotaxis as an experimental paradigm. In our example, we used six experimental conditions. Specifically, N2 wild-type C. elegans were conditioned on plates with low (0 mM), medium (50 mM), or high (100 mM) NaCl concentrations that were either seeded with the standard nematode food (E. coli OP50 bacterial strain) or left unseeded. Then, the C. elegans were allowed to roam an unseeded a...

To meaningfully interact with their environment and generate adaptive behavior, animals need to interpret the sensory information they receive: whether a given sensory cue is beneficial or detrimental to their survival is almost always not pre-determined but depends on the context. A key task of sensory and nervous systems is, therefore, to assign valence to environmental information through associative learning. Some stimuli are inherently appetitive, such as food, and others are inherently aversive, such as nociceptive cues. Such unconditional stimuli (US) can alter the responses to other stimuli presented together with them; as a result, animals can develop an attraction or aversion to the other, conditioned stimulus (CS). This is interpreted in such a way that CS signals or predicts the US1,2,3. Studying models of associative learning is necessary to elucidate the underlying genetic, cellular, and physiological mechanisms. However, this can be challenging due to the need to use in vivo models, the variability of behavior, the required licensing of animal handling, or the often-long timelines of training both the researcher and the animals, and this usually precludes it being carried out by undergraduate students in a research setting, and even more so in a teaching setting.
Therefore, invertebrate models offer the opportunity to conduct associative learning assays with undergraduate or MSc students. Invertebrates do not require animal licensing to handle them and can be cost-effective. A frequently used paradigm for associative learning is salt chemotaxis learning in C. elegans4,5,6,7,8,9.&#160;C. elegans sense the concentration of sodium chloride (NaCl) in their environment and learn to associate a particular [NaCl] with the presence or absence of food, respectively. Generally, animals learn to migrate towards salt concentrations that were previously presented with ample bacterial food and learn to migrate away from those that were presented in the absence of bacterial food. Salt is thus the conditioned stimulus, and food or starvation is the unconditional&#160;stimulus3. This association is formed within less than an hour and is an example of short-term associative memory. Associative learning of NaCl chemotaxis is modulated, amongst others, by calcium/calmodulin-dependent kinase&#160;(CaMK)&#160;signaling, dopaminergic and insulin/phosphatidylinositol (PI) 3-kinase signalling5,9,10, which suggests that it involves motivational systems and CaMK-dependent synaptic plasticity and may operate in a similar way as the signaling pathways governing associative learning in vertebrates.
In an established protocol for salt chemotaxis learning, animals migrate in a spatial gradient of salt on an agar plate created by placing two agar plugs with no or high salt, respectively, on the plate7. These assays are difficult to perform in a quantitative way by undergraduates due to the dynamic change in NaCl concentration on the chemotaxis assay plate over time and space, because the NaCl from the plugs continuously diffuses into the agar plate. An easier-to-perform assay is the use of quadrant plates, where two different salt concentrations (e.g., 25 mM NaCl and 0 mM NaCl) are presented and separated by plastic barriers11. However, because of the presentation of only two different NaCl concentrations, it cannot be used simultaneously to assay attraction to and/or avoidance of lowered or increased NaCl relative to the baseline condition. C. elegans is usually grown on NGM (Nematode Growth Media) plates in the presence of 50 mM NaCl and E. coli OP50 bacteria12, and therefore, forms a positive association of this NaCl concentration with food by default.
Here, we present an adapted salt chemotaxis learning protocol that can be performed by both researchers and undergraduates. Animals are first conditioned to different NaCl environments presented in the presence or absence of food. They are then transferred to assay plates with a choice of low, medium, and high salt concentrations in Y-chambered agar plates. Their chemotaxis learning in this setting can be used to investigate the effect of various factors modulating it, such as genotype, environmental contexts, or age of the animals.
This protocol is tailored to an undergraduate practical laboratory class split into eight student groups, with each student group performing a set of six measurements at each experimental time point (one measurement per experimental condition). However, the protocol can be adjusted to work for a single experimenter or for any number of groups/people, with appropriate changes to reagent quantities.

<table><tbody><tr><td>Agar</td><td>Chemsolute</td><td>9972-2KG</td><td></td></tr><tr><td>Bacto-peptone</td><td>Gibco</td><td>211677</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; 1M solution&amp;nbsp;</td><td>Merck</td><td>2389</td><td></td></tr><tr><td>&lt;em&gt;Caenorhabditis elegans&lt;/em&gt; strains</td><td>Caenorhabditis Genetics Center (CGC)</td><td></td><td></td></tr><tr><td>Cholesterol&amp;nbsp;</td><td>Roth</td><td>8866.1</td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; OP50 liquid culture</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Ethanol</td><td>Chemsolute</td><td>22731000</td><td></td></tr><tr><td>KH2PO4&amp;nbsp;</td><td>Roth</td><td>&amp;nbsp;P018.2</td><td></td></tr><tr><td>KPO&lt;sub&gt;4&lt;/sub&gt; (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4)&lt;/sub&gt; 1 M solution&amp;nbsp;</td><td>Chemsolute</td><td>16341000</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt; 1 M solution&amp;nbsp;</td><td>Roth</td><td>T888.2</td><td></td></tr><tr><td>Microcentrifuge tubes 1.5 mL</td><td>Eppendorf</td><td>N/A</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;</td><td>Roth</td><td>T877.1</td><td></td></tr><tr><td>NaCl</td><td>Chemsolute</td><td>13671000</td><td></td></tr><tr><td>Y-Petri dishes 90 x 10 mm</td><td>Moonlab</td><td>4-0033</td><td></td></tr></tbody></table>

measuring associative learning in chemotaxis of the nematode caenorhabditis elegans

Measuring Associative Learning in Chemotaxis of the Nematode Caenorhabditis elegans

To adapt their behavior, animals assign valence to sensory information through associative learning, and update this association when environmental conditions change. The behavioral responses of Caenorhabditis elegans to sodium chloride&#160;offer a well-defined paradigm for investigating the mechanisms governing associative learning. Exposing worms to specific NaCl levels paired with the presence or absence of food elicits chemotaxis towards or away from this NaCl concentration, respectively. The associative memory is formed within a few hours and can be modified in the same timeframe upon exposing the animals to a different NaCl concentration.
Here, we describe how to assay behavioral plasticity and learning based on C. elegans salt chemotaxis. The assay strains are grown in synchronized cohorts until they reach adulthood and are then conditioned for several hours on plates with different NaCl concentrations, either in the presence or absence of food. Animals are then transferred to assay plates that contain regions of low, medium, or high NaCl concentrations and allowed to crawl for 30 min before counting the number of worms in each region. A chemotaxis index is then calculated from the worms&#39; distribution on the plate. The assay can be used to test the effect of a range of factors affecting learning.

Conditioning for 4 h on plates with various amounts of NaCl (0 mM, 50 mM, 100 mM), with or without food, significantly affected the behavior of wild-type (N2) C. elegans. After 30 min on the assay plate, worms conditioned on plates with high NaCl concentrations and food (CP100+) and worms conditioned on plates with no NaCl and no food (CP0-) had a positive CI, indicating a preference for higher NaCl concentrations (Figure 2, Table 1, Table 2, and <s...

Here, we present a protocol to test associative learning by performing classical conditioning in the model organism C. elegans. The paradigm is based on pairing the concentration of salt in the environment with the presence or absence of food, which alters their chemotaxis towards or away from salt.

To adapt their behavior, animals assign valence to sensory information through associative learning, and update this association when environmental ...

One of the most fascinating questions in neuroscience is how learning memory emerge from the plasticity of neurons. Both plasticity and learning decline with age. But it is largely unclear why we investigate the mechanisms responsible for this decline.Using the nematode model organisms in neuritis elegans. It has become clear that nearly every behavior of C.elegans depends on context and experience, and that they share mechanisms of associative and non associative learning with other organisms. Remarkably key determinants of C.elegans learning such as neural activity and insulin signaling are also implicated in regulating the itching processes of the brain and even the entire organism.The basic technology is used in studying chemotaxis divided Petri dishes as through microscopes. We also use cameras with tracking software and custom built behavioral chambers for trajectory tracking and calcium imaging with microfluidics to visualize the physiological mechanisms behind the behaviors we observe on the Petri dish. Currently C.elegans sort chemotaxis Assays have been performed on either gradient plates or quadrant plates.Gradient plates can be demanding to perform because the gradient changes over time and quadrant plates, they have been used to compare two different concentrations of salt only. That's why we introduced the use of white plates for this Assay. We want to understand why the ability to learn, which depends on neural plasticity declines with age.We aim to use the chemotaxis assay to test whether specific signaling pathways are responsible for this decline. Based on this, we aim to develop treatments targeting this age-related cognitive decline. To begin obtain nematode growth, medium auger plates fed with live escherichia coli OP 50.Add Caenorhabditis elegans N two on the plate and maintain overnight at 20 degrees Celsius upside down to minimize moisture loss. The next day, transfer 30 young adult Caenorhabditis elegans onto the nematode growth plate. Allow animals to lay eggs for approximately eight hours at 20 degrees Celsius.After the timed egg laying, remove all adult worms from the plate. Culture the plate upside down at 20 degrees Celsius for three days. For the assay, seed half of the batch of the desired groups of conditioning plates with 200 microliters of OP 50 per plate.Allow the seeded plates to dry overnight at 20 degrees Celsius in an incubator alongside the unseeded plates. Prepare the desired concentration of one liter sodium chloride solution in a glass bottle. Autoclave the bottles for 15 minutes at 121 degrees Celsius.Then let the bottles cool for 30 minutes at 55 degrees Celsius in a water bath. Meanwhile, label the bottom of the YP tree dish section with the corresponding sodium chloride concentration, and draw a five millimeter radius circle around the plate center where the walls meet. Place each bottle one at a time on a heated magnetic stirer.Add 25 milliliters of one molar potassium phosphate solution at pH six to each bottle. Mix thoroughly for two minutes. Then sequentially add calcium chloride cholesterol, and magnesium sulfate while stirring.Next, aliquot the zero millimolar medium into one compartment of a Y Petri dish until it reaches the top of the internal wall. And let the medium solidify for approximately five minutes. Aliquot the 100 millimolar medium into the second compartment of the Y Petri dish until it touches the solidified zero millimolar section adjacent to it.Then use a sterile 10 microliter pipette tip to break the surface tension along the y wall and connect it to the zero millimolar sodium chloride section. Let the medium solidify for five minutes. Aliquot the 50 millimolar medium into the remaining compartment of the Y Petri dish until it touches the solidified zero millimolar and 100 millimolar sections.Then use a sterile 10 microliter pipette tip to break the surface tension along the Y wall and connect the 50 millimolar sodium chloride section to the other two sections. After letting the medium solidify for five minutes, store the plates overnight at 20 degrees Celsius. To begin pipette one milliliter of sterile M nine buffer onto a growth plate containing Caenorhabditis elegans.Swirl the plate gently to dislodge the worms from the OP 50 bacterial lawn and the auger forming a suspension. Tilt the plate to collect the worm suspension in M nine buffer using a one milliliter plastic pipette tip with the top part cutoff. Transfer the suspension to a 1.5 milliliter micro centrifuge tube.Allow the worms to settle at the bottom of the micro centrifuge tubes for approximately 60 seconds forming a pellet. Then remove the supernatant using a one milliliter pipette without disturbing the pellet. Wash the pellet with one milliliter of sterile M nine buffer twice.To remove residual OP 50. Reese suspend the pellet in 300 microliters of M nine buffer. After the final wash.Swirl the suspension for even distribution of worms and slowly transfer 50 microliters of worm suspension containing approximately 200 to 300 worms to each conditioning plate. For plates seeded with op 50, deposit the worm droplet away from the bacterial food lawn and let it get absorbed into the auger. Keep the plates upside down in an incubator at 20 degrees Celsius for four hours.After conditioning, pipette one milliliter of sterile M nine buffer onto a conditioning plate and swirl it gently to dislodge the worms forming a suspension. Then tilt the plate to collect the worm suspension using a one milliliter plastic pipette tip and transfer it to a 1.5 milliliter micro centrifuge tube. In 60 seconds, the worms settle down and form a pellet.After removing the supernatant, wash the worms two times with one milliliter of sterile M nine buffer. Then fill the micro centrifuge tube with M nine buffer up to the one milliliter mark. Once the worm pellet forms remove 960 microliters of the supernatant without disturbing the pellet.Now swirl or shake the tube gently to resuspend the worms. Transfer 20 microliters of this suspension containing approximately 100 to 150 worms to the center of an assay plate where the y wall segments meet. Let the worms roam for 30 minutes at 20 degrees Celsius.Then count the number of worms in each section of the assay plate and count them again after 30 minutes. Calculate the Chemotaxis index per plate per time point using the specified formula. Wild type Caenorhabditis elegans behavior was significantly affected by four hour conditioning on plates with different sodium chloride levels with or without food.After 30 minutes on the assay plate, worms conditioned with high sodium chloride concentrations and food, as well as those conditioned with no sodium chloride and no food showed a positive chemotaxis index indicating a preference for higher sodium chloride concentrations. Conversely, worms conditioned with high sodium chloride concentrations and no food, medium sodium chloride concentrations, and no food and no sodium chloride with food showed a negative chemotaxis index indicating a preference for lower sodium chloride concentrations. The Sodium chloride Food Association persisted after 60 minutes with the Chemotaxis index, often changing in worms conditioned with food indicating dynamic associative learning as animals update this association while roaming and searching for food.

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Research

JoVE Journal

Behavior

Here we describe methods to test C. elegans associative learning and short- and long-term associative memory. These population assays employ the worms  abilities to chemotax toward volatile odorants, and form positive associations upon pairing food with the chemoattractant butanone. Increasing the number of conditioning periods induces long-term memory.

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

This video introduces chemotaxis in the nematode C. elegans and shows a sample protocol testing chemotactic aversion to copper sulfate.

C. elegans Chemotaxis Assay: A Method to Test Chemosensation in Worms

Researchers can trigger on-demand movement in C. elegans by applying an electric field that worms sense and respond to.  This video introduces electrotactic behavior and shows a sample protocol that is done in a microfluidic device.

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

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.

C. elegans Chemotaxis Assay

We present a behavior recording setup and protocol that enables automated analysis of the nematode, Caenorhabditis elegans' preference for soluble compounds in a population-based assay. This article describes the construction of a behavior chamber, the behavioral assay protocol, and video analysis software usage.

Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

We have previously developed protocols for Caenorhabditis elegans to form short- and long-term associative memories by massed and spaced training, respectively. Here, detailed protocols are described for the conditioning of C. elegans by pairing 1-propanol and hydrochloric acid as conditioned and unconditioned stimuli, respectively, to form aversive associative memory.

Aversive Associative Learning and Memory Formation by Pairing Two Chemicals in Caenorhabditis elegans

This protocol establishes methods for extracting and quantifying responses to the volatile sex pheromone in C. elegans, providing tools to study chemical communication and navigation trajectory.

Volatile Sex Pheromone Extraction and Chemoattraction Assay in Caenorhabditis elegans

This protocol describes the modified hole board, which is a behavioral test set-up that comprises the characteristics of an open field and a traditional hole board. This set-up enables the differential analysis of unconditioned behavior of small laboratory mammals as well as the analysis of cognitive abilities.

The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats

A method is described in which 3-4 month old infants learn a task by discovery and their leg movements are captured to quantify the learning process.

Quantifying Learning in Young Infants: Tracking Leg Actions During a Discovery-learning Task

The Open-field Tower Maze (OFTM) is a maze created to study the behavioral and neural mechanisms of spatial learning (e.g., place- or response-learning) in rats. This maze is especially useful for experimenters who want to use a non-stressful maze paradigm to investigate spatial learning in their research.

Place and Response Learning in the Open-field Tower Maze

Learn classical conditioning protocols to measure associative learning and chemotaxis in Caenorhabditis elegans. This method reveals neural mechanisms underlying age-related cognitive decline.

Measuring Associative Learning in Chemotaxis of the Nematode Caenorhabditis elegans

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