Switch-like and persistent memory formation in individual Drosophila larvae

Associative learning allows animals to use past experience to predict future events. The circuits underlying memory formation support immediate and sustained changes in function, often in response to a single example. Larval Drosophila is a genetic model for memory formation that can be accessed at molecular, synaptic, cellular, and circuit levels, often simultaneously, but existing behavioral assays for larval learning and memory do not address individual animals, and it has been difficult to form long lasting memories, especially those requiring synaptic re-organization. We demonstrate a new assay for learning and memory capable of tracking the changing preferences of individual larvae. We use this assay to explore how activation of a pair of reward neurons changes the response to the innately aversive gas Carbon Dioxide (CO 2 ). We confirm that when coupled to odor presentation in appropriate temporal sequence, optogenetic reward reduces avoidance of CO 2 . We find that learning is switch-like: all-or-none and quantized in two states. Memories can be extinguished by repeated unrewarded exposure to CO 2 but are stabilized against extinction by repeated training or overnight consolidation. Finally, we demonstrate long-lasting protein synthesis dependent and independent memory formation.