Quantification of Appetitive and Mechanical Aversive Associative Learning Paradigms in Drosophila via a Y-maze Assay

Learning and memory are essential for the acquisition, storage, and recall of information, enabling organisms to adapt to changes in their environment. Associative learning begins when a stimulus, either positive or negative in valence, is presented alongside a neutral stimulus. This drives the development of an association between the stimuli, leading to the subsequent establishment of a learned behavioral response to the previously neutral stimulus. Associative learning is well conserved across species, from invertebrates to humans, and is highly amenable to experimental manipulation. Drosophila melanogaster is a valuable model for dissecting the mechanisms underlying associative learning and memory, given its genetic tractability, low cost, and the high-throughput nature of fly behavioral assays. Here, we describe two distinct associative learning paradigms in the Drosophila model that employ olfaction to establish short-term learned associations using a Y-maze assay. We first describe a novel negative associative learning paradigm employing a mechanical aversive stimulus. We next describe a positive associative learning paradigm in which flies learn to approach an odor paired with an appetitive sucrose reward. Overall, these protocols offer an inexpensive and streamlined approach to investigating associative learning under a multitude of different contexts. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Negative associative learning paradigm utilizing mechanical stimulation Basic Protocol 2: Positive associative learning paradigm utilizing sucrose feeding.