Animals approach stimuli that predict a pleasant outcome. After the paired presentation of an odour and a reward, Drosophila melanogaster can develop a conditioned approach towards that odour. Despite recent advances in understanding the neural circuits for associative memory and appetitive motivation, the cellular mechanisms for reward processing in the fly brain are unknown. Here we show that a group of dopamine neurons in the protocerebral anterior medial (PAM) cluster signals sugar reward by transient activation and inactivation of target neurons in intact behaving flies. These dopamine neurons are selectively required for the reinforcing property of, but not a reflexive response to, the sugar stimulus. In vivo calcium imaging revealed that these neurons are activated by sugar ingestion and the activation is increased on starvation. The output sites of the PAM neurons are mainly localized to the medial lobes of the mushroom bodies (MBs), where appetitive olfactory associative memory is formed. We therefore propose that the PAM cluster neurons endow a positive predictive value to the odour in the MBs. Dopamine in insects is known to mediate aversive reinforcement signals. Our results highlight the cellular specificity underlying the various roles of dopamine and the importance of spatially segregated local circuits within the MBs.

The mushroom body is required for a variety of behaviors of Drosophila melanogaster. Different types of intrinsic and extrinsic mushroom body neurons might underlie its functional diversity. There have been many GAL4 driver lines identified that prominently label the mushroom body intrinsic neurons, which are known as "Kenyon cells." Under one constant experimental condition, we analyzed and compared the the expression patterns of 25 GAL4 drivers labeling the mushroom body. As an internet resource, we established a digital catalog indexing representative confocal data of them. Further more, we counted the number of GAL4-positive Kenyon cells in each line. We found that approximately 2,000 Kenyon cells can be genetically labeled in total. Three major Kenyon cell subtypes, the gamma, alpha'/beta', and alpha/beta neurons, respectively, contribute to 33, 18, and 49% of 2,000 Kenyon cells. Taken together, this study lays groundwork for functional dissection of the mushroom body.

A paired presentation of an odor and electric shock induces aversive odor memory in Drosophila melanogaster. Electric shock reinforcement is mediated by dopaminergic neurons, and it converges with the odor signal in the mushroom body (MB). Dopamine is synthesized in approximately 280 neurons that form distinct cell clusters and is involved in a variety of brain functions. Recently, one of the dopaminergic clusters (PPL1) that includes MB-projecting neurons was shown to signal reinforcement for aversive odor memory. As each dopaminergic cluster contains multiple types of neurons with different projections and physiological characteristics, functional understanding of the circuit for aversive memory requires cellular identification. Here, we show that MB-M3, a specific type of dopaminergic neurons in the PAM cluster, is preferentially required for the formation of labile memory. Strikingly, flies formed significant aversive odor memory without electric shock when MB-M3 was selectively stimulated together with odor presentation. In addition, we identified another type of dopaminergic neurons in the PPL1 cluster, MB-MP1, which can induce aversive odor memory. As MB-M3 and MB-MP1 target the distinct subdomains of the MB, these reinforcement circuits might induce different forms of aversive memory in spatially segregated synapses in the MB.

Animals acquire predictive values of sensory stimuli through reinforcement. In the brain of Drosophila melanogaster, activation of two types of dopamine neurons in the PAM and PPL1 clusters has been shown to induce aversive odor memory. Here, we identified the third cell type and characterized aversive memories induced by these dopamine neurons. These three dopamine pathways all project to the mushroom body but terminate in the spatially segregated subdomains. To understand the functional difference of these dopamine pathways in electric shock reinforcement, we blocked each one of them during memory acquisition. We found that all three pathways partially contribute to electric shock memory. Notably, the memories mediated by these neurons differed in temporal stability. Furthermore, combinatorial activation of two of these pathways revealed significant interaction of individual memory components rather than their simple summation. These results cast light on a cellular mechanism by which a noxious event induces different dopamine signals to a single brain structure to synthesize an aversive memory.