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 ...

Two types of consolidated memory have been described in Drosophila, anesthesia-resistant memory (ARM), a shorter-lived form, and stabilized long-term memory (LTM). Until now, it has been thought that ARM and LTM coexist. On the contrary, we show that LTM formation leads to the extinction of ARM. Flies devoid of mushroom body vertical lobes cannot form LTM, but spaced conditioning can still erase their ARM, resulting in a remarkable situation: The more these flies are trained, the less they remember. We propose that ARM acts as a gating mechanism that ensures that LTM is formed only after repetitive and spaced training.

The neuromodulatory function of dopamine (DA) is an inherent feature of nervous systems of all animals. To learn more about the function of neural DA in Drosophila, we generated mutant flies that lack tyrosine hydroxylase, and thus DA biosynthesis, selectively in the nervous system. We found that DA is absent or below detection limits in the adult brain of these flies. Despite this, they have a lifespan similar to WT flies. These mutants show reduced activity, extended sleep time, locomotor deficits that increase with age, and they are hypophagic. Whereas odor and electrical shock avoidance are not affected, aversive olfactory learning is abolished. Instead, DA-deficient flies have an apparently "masochistic" tendency to prefer the shock-associated odor 2 h after conditioning. Similarly, sugar preference is absent, whereas sugar stimulation of foreleg taste neurons induces normal proboscis extension. Feeding the DA precursor L-DOPA to adults substantially rescues the learning deficit as well as other impaired behaviors that were tested. DA-deficient flies are also defective in positive phototaxis, without alteration in visual perception and optomotor response. Surprisingly, visual tracking is largely maintained, and these mutants still possess an efficient spatial orientation memory. Our findings show that flies can perform complex brain functions in the absence of neural DA, whereas specific behaviors involving, in particular, arousal and choice require normal levels of this neuromodulator.neurotransmitters | locomotor activity | memory formation | choice behavior | feeding behavior A n important challenge in neuroscience is to understand the roles of specific neurotransmitter systems on brain homeostasis and functioning. Dopamine (DA), a biogenic amine biosynthesized from tyrosine, is an essential neuromodulator in the mammalian central nervous system that is involved in attention, movement control, motivation, and cognition. Studies in Drosophila melanogaster indicate that DA also plays central regulatory roles in insects, specifically in the neural networks controlling locomotor activity and stereotypical behaviors (1-3), sleep and arousal (4-7), registration of salient stimuli (4,8,9), and associative olfactory learning (10-15). Some of these studies were based on genetic inactivation or overactivation of dopaminergic neurons. Dopaminergic neurons can corelease other neuroactive agents, such as neuropeptides, however. Therefore, one must ensure that the behavioral phenotypes observed specifically result from the lack of DA release to draw firm conclusions on brain DA function.Nearly all neuropil regions of the insect CNS receive dense dopaminergic innervation. In particular, the Drosophila adult brain contains six paired clusters of dopaminergic neurons, some of which specifically project to higher brain centers, such as the central complex and the mushroom bodies (1,10,12,13,(16)(17)(18). Tyrosine hydroxylase (TH) catalyzes the first and rate-limiting step in DA biosynthesis (Fig. S1A). Because DA is also ...

Adipokinetic hormone (AKH) is a metabolic neuropeptide principally known for its mobilization of energy substrates, notably lipid and trehalose during energy-requiring activities, such as flight and locomotion. Drosophila melanogaster AKH cell localization in corpora cardiaca, as in other insect species, was confirmed by immunoreactivity and by a genetic approach using the UAS/GAL4 system. To assess AKH general physiological rules, we ablated AKH endocrine cells by specifically driving the expression of apoptosis transgenes in AKH cells. Trehalose levels were decreased in larvae and starved adults, when the stimulation by AKH of the production of trehalose from fat body glycogen is no longer possible. Moreover, we show that these adults without AKH cells become progressively hypoactive. Finally, under starvation conditions, those hypoactive AKH-knockout cell flies survived approximately 50% longer than control wild-type flies, suggesting that the slower rate at which AKH-ablated flies mobilize their energy resources extends their survival.

Efficient energy use has constrained the evolution of nervous systems. However, it is unresolved whether energy metabolism may resultantly regulate major brain functions. Our observation that Drosophila flies double their sucrose intake at an early stage of long-term memory formation initiated the investigation of how energy metabolism intervenes in this process. Cellular-resolution imaging of energy metabolism reveals a concurrent elevation of energy consumption in neurons of the mushroom body, the fly's major memory centre. Strikingly, upregulation of mushroom body energy flux is both necessary and sufficient to drive long-term memory formation. This effect is triggered by a specific pair of dopaminergic neurons afferent to the mushroom bodies, via the D5-like DAMB dopamine receptor. Hence, dopamine signalling mediates an energy switch in the mushroom body that controls long-term memory encoding. Our data thus point to an instructional role for energy flux in the execution of demanding higher brain functions.