Measuring Associative Learning in Chemotaxis of the Nematode Caenorhabditis elegans

Summary

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.

Abstract

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 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' distribution on the plate. The assay can be used to test the effect of a range of factors affecting learning.

Introduction

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 US^1,2,3. 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. elegans^4,5,6,7,8,9. 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 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 (CaMK) signaling, dopaminergic and insulin/phosphatidylinositol (PI) 3-kinase signalling^5,9,10, 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.

Protocol

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.

1. Prepare four clean 500 mL glass bottles and add the following:
   1. 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);
   2. 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);
   3. 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).
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.

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.

1. 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.
2. Transfer 30 young (day 2) N2 adults onto each GP.
3. Allow animals to lay eggs for approximately 8 h at 20 °C.
4. Remove all adults from the plates and culture upside down at 20 °C for 3 days.

3. Preparation of assay (1 day before assay)

1. Prepare seeded (+) and unseeded (-) CP.
   1. 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 (<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.
   2. Allow plates to dry overnight at 20 °C in an incubator alongside the unseeded half of the CP (CP0-, CP50-, and CP100-, respectively)
2. Prepare assay plates (AP).
   1. Prepare three clean 1 L glass bottles and add the following:
      1. 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)
      2. 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)
      3. 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)
   2. 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.
   3. 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.
   4. 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.
   5. 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.
   6. 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.
   7. 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.
   8. Store the plates overnight at 20 °C in an incubator.
      NOTE: Approximately 50 AP can be made from this method.
3. Prepare 1 L of sterile M9 buffer¹⁴.

4. C. elegans associative learning assay (here performed by 8 groups in parallel)

1. 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.
2. 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.
3. 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.
4. Remove the supernatant/excess M9 buffer using a 1 mL pipette, taking care not to disturb the pellet.
5. 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.
6. 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.
7. 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.
8. Pipette 1 mL of sterile M9 buffer onto a CP and swirl the plate gently to dislodge the worms, forming a suspension.
9. 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.
10. Allow the worms to settle to the bottom of the microcentrifuge tube for approximately 60 s.
11. Remove the supernatant/excess M9 buffer using a 1 mL pipette without disturbing the worm pellet.
12. Wash the worms two further times with 1 mL of sterile M9 buffer. Allow the pellet to settle for approximately 60 s between washes.
13. Fill the microcentrifuge tube with M9 buffer to the 1 mL mark and allow the worm pellet to settle for approximately 60 s.
14. Remove 960 µL of the supernatant using a 1 mL pipette, taking care not to disturb the pellet.
15. 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.
16. 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).
17. 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.
18. Calculate the chemotaxis index (CI) per plate per time point using the formula^15,16:
    

Results

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

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agar	Chemsolute	9972-2KG
Bacto-peptone	Gibco	211677
CaCl2 1M solution	Merck	2389
Caenorhabditis elegans strains	Caenorhabditis Genetics Center (CGC)
Cholesterol	Roth	8866.1
E. coli OP50 liquid culture	N/A	N/A
Ethanol	Chemsolute	22731000
KH2PO4	Roth	P018.2
KPO4 (KH2PO4, K2HPO4) 1 M solution	Chemsolute	16341000
MgSO4 1 M solution	Roth	T888.2
Microcentrifuge tubes 1.5 mL	Eppendorf	N/A
Na2HPO4	Roth	T877.1
NaCl	Chemsolute	13671000
Y-Petri dishes 90 x 10 mm	Moonlab	4-0033