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 attr....

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);

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