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.

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 ...

A fundamental duty of any efficient memory system is to prevent long-lasting storage of poorly relevant information. However, little is known about dedicated mechanisms that appropriately trigger production of long-term memory (LTM). We examined the role of Drosophila dopaminergic neurons in the control of LTM formation and found that they act as a switch between two exclusive consolidation pathways leading to LTM or anesthesia-resistant memory (ARM). Blockade, after aversive olfactory conditioning, of three pairs of dopaminergic neurons projecting on mushroom bodies, the olfactory memory center, enhanced ARM, whereas their overactivation conversely impaired ARM. Notably, blockade of these neurons during the intertrial intervals of a spaced training precluded LTM formation. Two pairs of these dopaminergic neurons displayed sustained calcium oscillations in naive flies. Oscillations were weakened by ARM-inducing massed training and were enhanced during LTM formation. Our results indicate that oscillations of two pairs of dopaminergic neurons control ARM levels and gate LTM.

structure ͉ cell growth T he insulin-like growth factor-binding protein (IGFBP) family comprises six soluble proteins (IGFBP1-6) of Ϸ250 residues that bind to IGFs with nanomolar affinities (1-4). Because of their sequence homology, IGFBPs are assumed to share a common overall fold and are expected to have closely related IGF-binding determinants. Each IGFBP can be divided into three distinct domains of approximately equal lengths: highly conserved cysteinerich N and C domains and a central linker domain unique to each IGFBP species. Both the N and C domains participate in the binding to IGFs, although the specific roles of each of these domains in IGF binding have not been decisively determined (1-13). The C-terminal domain may be responsible for preferences of IGFBPs for one species of IGF over the other (2, 3-7, 9-13); the C-terminal domain is also involved in regulation of the IGF-binding affinity through interaction with extracellular matrix components (1,2,14) and is most probably engaged in mediating IGF1-independent actions (1, 4, 14). The central linker domain is the least conserved region and has never been cited as part of the IGF-binding site for any IGFBP. This domain is the site of posttranslational modifications, specific proteolysis (4), and the acid-labile subunit (1) and extracellular matrix associations (1, 2, 14) known for IGFBPs. Proteolytic cleavage in this domain is believed to produce loweraffinity N-and C-terminal fragments that cannot compete with IGF receptors for IGFs, and, thus, the proteolysis is assumed to be the predominant mechanism for IGF release from IGFBPs (4, 9).However, recent studies indicate that the resulting N-and Cterminal fragments still can inhibit IGF activity and have functional properties that differ from those of the intact proteins (1, 3, 5, 9).The structure of the N-terminal domain of IGFBP-5, free (15) and complexed to IGF1 (16), was solved some time ago. More recently, low-resolution structures of the C-terminal domain of IGFBP6 (12) and its binding surface on IGF2 (3, 12) have been determined with NMR spectroscopy. There is, however, no x-ray structure of a ternary complex of the C-terminal domain of any IGFBPs bound to both the N-terminal domain and IGF, although the C-terminal fragment of IGFBP4 was crystallized recently (9), and also the x-ray structure of the isolated C-terminal fragment of IGFBP1 has been solved (17). We recently reported the x-ray structure of the ternary complex of the N-and C-terminal domains of IGFBP4 bound to IGF1 (10) and described ordered structures for the N-terminal domain of IGFBP4 and IGF1. The C domain was represented by disconnected patches of electron density and could not be interpreted. We describe here the long-sought, highresolution x-ray structure of a complex of the N-and C-terminal domains of IGFBP4 bound to IGF1. We also present the structure of the C-terminal domain of IGFBP1 bound to the N-terminal domain of IGFBP4 and IGF1 and the structure of the binary complex of the N-terminal domain of IGFBP4 (residues 1-92)...

The amphipod crustacean Parhyale hawaiensis is a blossoming model system for studies of developmental mechanisms and more recently regeneration. We have sequenced the genome allowing annotation of all key signaling pathways, transcription factors, and non-coding RNAs that will enhance ongoing functional studies. Parhyale is a member of the Malacostraca clade, which includes crustacean food crop species. We analysed the immunity related genes of Parhyale as an important comparative system for these species, where immunity related aquaculture problems have increased as farming has intensified. We also find that Parhyale and other species within Multicrustacea contain the enzyme sets necessary to perform lignocellulose digestion ('wood eating'), suggesting this ability may predate the diversification of this lineage. Our data provide an essential resource for further development of Parhyale as an experimental model. The first malacostracan genome will underpin ongoing comparative work in food crop species and research investigating lignocellulose as an energy source.DOI: http://dx.doi.org/10.7554/eLife.20062.001