6 These authors contributed equally to this work. 2 Aversive olfactory memory is formed in the Drosophila mushroom bodies (MB).Memory retrieval requires MB output, but it remains unknown how a memory trace in the MB drives conditioned avoidance of a learned odour. To identify neurons involved in olfactory memory retrieval, we performed an anatomical and functional screen of defined sets of MB extrinsic neurons. Here we show that MB-V2 neurons are essential for retrieval of both short-and long-lasting memory, but neither for memory formation nor for memory consolidation. We further show that MB-V2 are cholinergic efferent neurons that project from the MB vertical lobes to the middle superiormedial protocerebrum and the lateral horn (LH). Notably, the odour response of MB-V2 neurons is modified after conditioning. As the LH is implicated in innate responses to repellent odorants, we propose that during memory retrieval, MB-V2 neurons reinforce the olfactory pathway involved in innate odour avoidance.Different odours induce innate approach or avoidance behaviours in Drosophila. Innate odour responses can be modulated by experience, such as associative learning. After simultaneous exposure to an electric shock and an odorant, flies form aversive memory and show robust conditioned odour avoidance that lasts for hours to days, depending on the training protocol [1][2][3] . The neural pathways for odour or shock processing and signal integration in the fly brain have been intensely studied in recent years. Odour information is first represented in the antennal lobes in the form of olfactory receptor neuron activity 4 . Projection neurons then convey this information to higher order processing centres 4 : the mushroom bodies (MB) and the lateral horn (LH). In contrast, aversive reinforcement signals in response to electric shock are relayed to the MB via dopaminergic neurons [5][6][7] . The olfactory and 3 electric shock signals are integrated in the MB to form aversive olfactory memory 1, 2 . The MB are however dispensable for innate avoidance of the repellent odours 8,9 .In adult Drosophila, the MB consist of approximately 2000 Kenyon cells per brain hemisphere, which may be classified into three major types based on their axonal projection: γ neurons, which form only a medial lobe, α/β neurons, whose axons branch to form a vertical (α) and a medial (β) lobe, and α'/β' neurons, which also form a vertical (α') and a medial (β') lobe 10 . Functional brain imaging has revealed localised activation of cAMP/PKA signalling in the MB α lobe in response to simultaneous cholinergic and dopaminergic stimulation 11,12 , that represent respectively the odorant and electric shock pathways.Following associative conditioning, calcium imaging studies have shown that a short-term memory trace is formed in the α'/β' neurons 13, and a long-term one in α lobes 14 . Previous studies have shown that the output of the α/β neurons is necessary for the retrieval of all phases of olfactory memory 15,16 , but the neural circuits that translate ...