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

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.

Learning and memory are vital for animals to survive and reproduce by efficiently navigating ever-changing environments. It's also one of the major subjects studied in neuroscience. Caenorhabditis elegans is an attractive organism for the study of learning and memory because of the simplicity of its nervous system.

Its chemical and electrical wiring diagrams are also completely reconstructed. Caenorhabditis elegans'behavioral analysis is extremely tricky and is affected by a number of parameters such as chemical, mechanical and temperature stresses. To begin, prepare six centimeter nematode growth medium, or NGM plates, by spreading 0.2 milliliters of an E.coli OP50 liquid culture in a Luria-Bertani medium and incubating them at room temperature for no more than 24 hours.

On day one, pick and place five well fed gravid animals on each six centimeter NGM plate with a platinum worm picker and let them lay around 50 eggs for three hours at room temperature to obtain a synchronized population of adult animals. To stop egg laying, remove parent animals from the plates with a platinum worm picker. Cultivate the animals at room temperature for about five days to reach the mature adult stage, not the young adult stage.

After five days, collect around 200 adult animals in an animal collector by washing each plate with one milliliter of 0.25%aqueous gelatin which will prevent the adhesion of the animals to the surface of plastics, such as pipette tips. Then, wash the animals by gently moving the collector up and down twice in 10 milliliters of double distilled water. Repeat the process twice.

Gently immerse the animal collector containing around 200 animals in 40 milliliters of a mixture of 1%1-propanol and hydrochloric acid at pH 4 in a crystallizing dish for one second. Wash the animals in the collector by very gently immersing the collector once in a well of its 6-well tissue culture plate containing 10 milliliters of double distilled water. Then place the collector on an E.coli OP50 lawn on a six centimeter NGM plate for 10 minutes for the animals to rest.

Next, wash the animals in the collector with 10 milliliters of double distilled water by gently moving the collector up and down. Repeat this wash thrice and proceed to chemotaxis assay. Sequentially wash the animals in a collector with propanol mixture and double distilled water, once each as demonstrated earlier.

Next place the collector on an E.coli OP50 lawn on an NGM agar in a six centimeter Petri dish for 10 minutes for the animals to rest. Next, wash the animals thrice by gently moving the collector up and down twice in 10 milliliters of double distilled water to prevent bacterial contamination and proceed to chemotaxis assay. Prepare auger plates for the chemotaxis assay in six centimeter plastic Petri dishes.

Then use a sawed-off pipette tip having an opening of more than one millimeter to transfer the animals from the collector to a two milliliter micro centrifuge tube containing one milliliter of 0.25%aqueous gelatin. After the animals settle at the bottom of the tube by gravity, remove the supernatant from the tube. Resuspend the animals in one milliliter of chemotaxis assay buffer, and let them settle down by gravity to the bottom of the tube for around one minute.

Then, remove as much supernatant as possible by pipetting. Next, diagonally spot four microliters of 5%aqueous 1-propanol at two places, then spot four microliters of double distilled water at two other places in the Petri plate. Then use a sawed-off pipette tip with a one millimeter opening to spot six microliters of the animal suspension in a chemotaxis assay buffer containing around 60 animals at the center of three plates.

Remove the liquid as much as possible with a laboratory tissue wick without touching the animals. Place a lid on the plate and allow the animals to move freely on the plate for 10 minutes at room temperature. To stop the chemotaxis, transfer this plate into a glass Petri dish on ice for three minutes.

Next, place the plate in a refrigerator until counting the number of animals on the plate. Count the number of animals in four sections except for those in the center circle under a stereo microscope, and calculate the chemotaxis index using the equation. From the chemotaxis index values, calculate learning index values as the difference between the chemotaxis index value of the reference animals and the chemotaxis index value of the conditioned animals.

In the chemotaxis assay, animals conditioned with the mixture of 1-propanol and hydrochloric acid we're not attracted to 5%1-propanol spotted on agar plates, whereas naive and reference animals were attracted to 5%1-propanol. Animals conditioned by the spaced training with a mixture of aqueous 1-propanol and hydrochloric acid were no longer attracted to 5%1-propanol compared to animals treated with 1%1-propanol or hydrochloric acid, or double distilled water only. Studying the effect of mutations on memory revealed that short-term and long-term memory formation depended on the genes playing essential roles in the classical conditioning of the animals.

After each conditioning, animals should be gently washed only once with double distilled water. After long-term memory is formed using the procedure, calcium imaging and electrophysiology can identify neuronal circuits responsible for the memory. Through genetic and single neuron analyses, the memory storage and retrieval processes can be addressed at molecular and single cell levels.