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

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment through neuron-specific detection of food and chemical odors(1, 2), and can associate nutritive states with chemical odors(3), temperature(4), and the pathogenicity of a food source(5). Here, we describe assays of C. elegans associative learning and short-and long-term associative memory. We modified an aversive olfactory learning paradigm(6) to instead produce a positive response; the assay involves starving similar to 400 worms, then feeding the worms in the presence of the AWC neuron-sensed volatile chemoattractant butanone at a concentration that elicits a low chemotactic index (similar to Toroyama et al.(7)). A standard population chemotaxis assay1 tests the worms' attraction to the odorant immediately or minutes to hours after conditioning. After conditioning, wild-type animals' chemotaxis to butanone increases similar to 0.6 Chemotaxis Index units, its "Learning Index". Associative learning is dependent on the presence of both food and butanone during training. Pairing food and butanone for a single conditioning period ("massed training") produces short-term associative memory that lasts similar to 2 hours. Multiple conditioning periods with rest periods between ("spaced training") yields long-term associative memory (<40 hours), and is dependent on the cAMP Response Element Binding protein ( CREB), 6 a transcription factor required for long-term memory across species.(8) Our protocol also includes image analysis methods for quick and accurate determination of chemotaxis indices. High-contrast images of animals on chemotaxis assay plates are captured and analyzed by worm counting software in MatLab. The software corrects for uneven background using a morphological tophat transformation.(9) Otsu's method is then used to determine a threshold to separate worms from the background.(10) Very small particles are removed automatically and larger non-worm regions (plate edges or agar punches) are removed by manual selection. The software then estimates the size of single worm by ignoring regions that are above a specified maximum size and taking the median size of the remaining regions. The number of worms is then estimated by dividing the total area identified as occupied by worms by the estimated size of a single worm. We have found that learning and short-and long-term memory can be distinguished, and that these processes share similar key molecules with higher organisms.(6,8) Our assays can quickly test novel candidate genes or molecules that affect learning and short-or long-term memory in C. elegans that are relevant across species.