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Predicting positive or negative reinforcement from environmental clues is essential to guide decision making and goal-directed behavior. In insect brains the mushroom body is a central structure for learning such valuable associations between sensory signals and reinforcement. We propose a biologically realistic spiking network model of theDrosophilalarval olfactory pathway for the association of odors and reinforcement to bias behavior towards either approach or avoidance. We demonstrate that prediction error coding through integration of present and expected reinforcement in dopaminergic neurons can serve as a driving force in learning that can, combined with synaptic homeostasis, account for the experimentally observed features of acquisition and extinction of associations that depend on the intensity of odor and reward, as well as temporal features of the odor/reward pairing. To allow for a direct comparison of our simulation results with behavioral data we model learning-induced plasticity over the full time course of behavioral experiments and simulate locomotion of individual larvae towards or away from odor sources in a virtual environment.

Larvae of the fruit flyDrosophila melanogasterare a powerful study case for understanding the neural circuits underlying behavior. Indeed, the numerical simplicity of the larval brain has permitted the reconstruction of its synaptic connectome, and genetic tools for manipulating single, identified neurons allow neural circuit function to be investigated with relative ease and precision. We focus on one of the most complex neurons in the brain of the larva (of either sex), the GABAergic anterior paired lateral neuron (APL). Using behavioral and connectomic analyses, optogenetics, Ca2+imaging and pharmacology, we study how APL affects associative olfactory memory. We first provide a detailed account of the structure, regional polarity, connectivity, and metamorphic development of APL, and further confirm that optogenetic activation of APL has an inhibiting effect on its main targets, the mushroom body Kenyon cells. All these findings are consistent with the previously identified function of APL in the sparsening of sensory representations. To our surprise, however, we found that optogenetically activating APL can also have a strong rewarding effect. Specifically, APL activation together with odor presentation establishes an odor-specific, appetitive, associative short-term memory, whereas naïve olfactory behavior remains unaffected. An acute, systemic inhibition of dopamine synthesis as well as an ablation of the dopaminergic pPAM neurons impair reward learning through APL activation. Our findings provide a study case of complex circuit function in a numerically simple brain, and suggest a previously unrecognized capacity of central-brain GABAergic neurons to engage in dopaminergic reinforcement.Significance statementThe single, identified giant anterior paired lateral (APL) neuron is one of the most complex neurons in the insect brain. It is GABAergic and contributes to the sparsening of neuronal activity in the mushroom body, the memory center of insects. We provide the most detailed account yet of the structure of APL in larvalDrosophilaas a neurogenetically accessible study case. We further reveal that, contrary to expectations, the experimental activation of APL can exert a rewarding effect, likely via dopaminergic reward pathways. The present study both provides an example of unexpected circuit complexity in a numerically simple brain, and reports an unexpected effect of activity in central-brain GABAergic circuits.

Memory scores are dynamic across developmental stages. In particular, memory scores typically decrease from late adolescence into old age, reflecting complex changes in mnemonic and sensory-motor faculties, metabolic and motivational changes, and changes in cognitive strategy as well. In Drosophila melanogaster, such age-related decreases in memory scores have been studied intensely for the association of odours with electric shock punishment. We report that odour-sucrose reward memory scores likewise decrease as the flies age. This was observed after one-trial and after two-trial conditioning, and for both immediate testing and for recall tests one day later. This decrease was already particularly pronounced in relatively young animals, in the first 2-3 weeks after adult hatching, and was more pronounced in female than in male flies.

Neuronally orchestrated muscular movement and locomotion are defining faculties of multicellular animals. Due to its numerically simple brain and neuromuscular system and its genetic accessibility, the larva of the fruit fly Drosophila melanogaster is an established model to study these processes at tractable levels of complexity. However, although the faculty of locomotion clearly pertains to the individual animal, present studies of locomotion in larval Drosophila mostly use group assays and measurements aggregated across individual animals. The alternative is to measure animals one at a time, an extravagance for larger-scale analyses. In principle or in practice, this in particular rules out grasping the inter- and intra-individual variability in locomotion and its genetic and neuronal determinants. Here we present the IMBA (Individual Maggot Behaviour Analyser) for tracking and analysing the behaviour of individual larvae within groups. Using a combination of computational modelling and statistical approaches, the IMBA reliably resolves individual identity across collisions. It does not require specific hardware and can therefore be used in non-expert labs. We take advantage of the IMBA first to systematically describe the inter- and intra-individual variability in free, unconstrained locomotion in wild-type animals. We then report the discovery of a novel, complex locomotion phenotype of a mutant lacking an adhesion-type GPCR. The IMBA further allows us to determine, at the level of individual animals, the modulation of locomotion across repeated activations of dopamine neurons. Strikingly, IMBA can also be used to analyse ‘silly walks’, that is patterns of locomotion it was not originally designed to investigate. This is shown for the transient backward locomotion induced by brief optogenetic activation of the brain-descending ‘mooncrawler’ neurons, and the variability in this behaviour. Thus, the IMBA is an easy-to-use toolbox allowing an unprecedentedly rich view of the behaviour and behavioural variability of individual Drosophila larvae, with utility in multiple biomedical research contexts.

Larval Drosophila are used as a genetically accessible study case in many areas of biological research. Here we report a fast, robust and user-friendly procedure for the whole-body multifluorescence imaging of Drosophila larvae; the protocol has been optimized specifically for larvae by systematically tackling the pitfalls associated with clearing this small but cuticularized organism. Tests on various fluorescent proteins reveal that the recently introduced monomeric infrared fluorescent protein (mIFP) is particularly suitable for our approach. This approach comprises an effective, low-cost clearing protocol with minimal handling time and reduced toxicity in the reagents employed. It combines a success rate high enough to allow for small-scale screening approaches and a resolution sufficient for cellular-resolution analyses with light sheet and confocal microscopy. Given that publications and database documentations typically specify expression patterns of transgenic driver lines only within a given organ system of interest, the present procedure should be versatile enough to extend such documentation systematically to the whole body. As examples, the expression patterns of transgenic driver lines covering the majority of neurons, or subsets of chemosensory, central brain or motor neurons, are documented in the context of whole larval body volumes (using nsyb-Gal4, IR76b-Gal4, APL-Gal4 and mushroom body Kenyon cells, or OK371-Gal4, respectively). Notably, the presented protocol allows for triple-color fluorescence imaging with near-infrared, red and yellow fluorescent proteins.

Humans and animals alike form oppositely valenced memories for stimuli that predict the occurrence versus the termination of a reward: appetitive ‘reward’ memory for stimuli associated with the occurrence of a reward and aversive ‘frustration’ memory for stimuli that are associated with its termination. We characterize these memories in larval Drosophila using a combination of Pavlovian conditioning, optogenetic activation of the dopaminergic central-brain DAN-i1864 neuron, and high-resolution video-tracking. This reveals their dependency on the number of training trials and the duration of DAN-i1864 activation, their temporal stability, and the parameters of locomotion that are modulated during memory expression. Together with previous results on ‘punishment’ versus ‘relief’ learning by DAN-f1 neuron activation, this reveals a 2×2 matrix of timing-dependent memory valence for the occurrence/ termination of reward/ punishment. These findings should aid the understanding and modelling of how brains decipher the predictive, causal structure of events around a target reinforcing occurrence.

Animals, including humans, form oppositely valenced memories for stimuli that predict the occurrence versus the termination of a reward: appetitive ‘reward’ memory for stimuli associated with the occurrence of a reward and aversive ‘frustration’ memory for stimuli that are associated with its termination. We characterized these memories in larval Drosophila melanogaster using a combination of Pavlovian conditioning, optogenetic activation of the dopaminergic central-brain DAN-i1864 neuron, and high-resolution video-tracking. This reveals their dependency on the number of training trials and the duration of DAN-i1864 activation, their temporal stability, and the parameters of locomotion that are modulated during memory expression. Together with previous results on ‘punishment’ versus ‘relief’ learning by DAN-f1 neuron activation, this reveals a 2×2 matrix of timing-dependent memory valence for the occurrence/termination of reward/punishment. These findings should aid the understanding and modelling of how brains decipher the predictive, causal structure of events around a target reinforcing occurrence.

Animals of many species are capable of “small data” learning, that is, of learning without repetition. Here we introduce larval Drosophila melanogaster as a relatively simple study case for such one-trial learning. Using odor-food associative conditioning, we first show that a sugar that is both sweet and nutritious (fructose) and sugars that are only sweet (arabinose) or only nutritious (sorbitol) all support appetitive one-trial learning. The same is the case for the optogenetic activation of a subset of dopaminergic neurons innervating the mushroom body, the memory center of the insects. In contrast, no one-trial learning is observed for an amino acid reward (aspartic acid). As regards the aversive domain, one-trial learning is demonstrated for high-concentration sodium chloride, but is not observed for a bitter tastant (quinine). Second, we provide follow-up, parametric analyses of odor-fructose learning. Specifically, we ascertain its dependency on the number and duration of training trials, the requirements for the behavioral expression of one-trial odor-fructose memory, its temporal stability, and the feasibility of one-trial differential conditioning. Our results set the stage for a neurogenetic analysis of one-trial learning and define the requirements for modeling mnemonic processes in the larva.

The Drosophila larva is a relatively simple, 10 000-neuron study case for learning and memory with enticing analytical power, combining genetic tractability, the availability of robust behavioral assays, the opportunity for single-cell transgenic manipulation, and an emerging synaptic connectome of its complete central nervous system. Indeed, although the insect mushroom body is a much-studied memory network, the connectome revealed that more than half of the classes of connection within the mushroom body had escaped attention. The connectome also revealed circuitry that integrates, both within and across brain hemispheres, higher-order sensory input, intersecting valence signals, and output neurons that instruct behavior. Further, it was found that activating individual dopaminergic mushroom body input neurons can have a rewarding or a punishing effect on olfactory stimuli associated with it, depending on the relative timing of this activation, and that larvae form molecularly dissociable short-term, long-term, and amnesia-resistant memories. Together, the larval mushroom body is a suitable study case to achieve a nuanced account of molecular function in a behaviorally meaningful memory network.

Dopamine serves many functions, and dopamine neurons are correspondingly diverse. We use a combination of optogenetics, behavioral experiments, and high-resolution video-tracking to probe for the functional capacities of two single, identified dopamine neurons in larval Drosophila. The DAN-f1 and the DAN-d1 neuron were recently found to carry aversive teaching signals during Pavlovian olfactory learning. We enquire into a fundamental feature of these teaching signals, namely their temporal "fingerprint". That is, receiving punishment feels bad, whereas being relieved from it feels good, and animals and humans alike learn with opposite valence about the occurrence and the termination of punishment (the same principle applies in the appetitive domain, with opposite sign). We find that DAN-f1 but not DAN-d1 can mediate such timing-dependent valence reversal: presenting an odor before DAN-f1 activation leads to learned avoidance of the odor (punishment memory), whereas presenting the odor upon termination of DAN-f1 activation leads to learned approach (relief memory). In contrast, DAN-d1 confers punishment memory only. These effects are further characterized in terms of the impact of the duration of optogenetic activation, the temporal stability of the memories thus established, and the specific microbehavioral patterns of locomotion through which they are expressed. Together with recent findings in the appetitive domain and from adult Drosophila, our results suggest that heterogeneity in the temporal fingerprint of teaching signals might be a more general principle of reinforcement processing through dopamine neurons.

Preparations of Rhodiola rosea root are widely used in traditional medicine. They can increase life span in worms and flies, and have various effects related to nervous system function in different animal species and humans. However, which of the compounds in R. rosea is mediating any one of these effects has remained unknown in most cases. Here, an analysis of the volatile and non-volatile low-molecular-weight constituents of R. rosea root samples was accompanied by an investigation of their behavioral impact on Drosophila melanogaster larvae. Rhodiola rosea root samples have an attractive smell and taste to the larvae, and exert a rewarding effect. This rewarding effect was also observed for R. rosea root extracts, and did not require activity of dopamine neurons that mediate known rewards such as sugar. Based on the chemical profiles of R. rosea root extracts and resultant fractions, a bioactivity-correlation analysis (AcorA) was performed to identify candidate rewarding compounds. This suggested positive correlations for – among related compounds – ferulic acid eicosyl ester (FAE-20) and β-sitosterol glucoside. A validation using these as pure compounds confirmed that the correlations were causal. Their rewarding effects can be observed even at low micromolar concentrations and thus at remarkably lower doses than for any known taste reward in the larva. We discuss whether similar rewarding effects, should they be observed in humans, would indicate a habit-forming or addictive potential.

This special issue is dedicated to ‘Future Perspectives of Neurogenetics’ in honor of Troy D. Zars (1967–2018). It is intended to offer insight and inspiration for our understanding of how adaptive...

Preparations of

Troy D. Zars (1967–2018) was an American biologist. He studied the relationships between genes, neuronal circuits and behavior in the fruit fly Drosophila melanogaster. Zars co-pioneered the use of...

Associating stimuli with positive or negative reinforcement is essential for survival, but a complete wiring diagram of a higher-order circuit supporting associative memory has not been previously available. Here we reconstruct one such circuit at synaptic resolution, the Drosophila larval mushroom body. We find that most Kenyon cells integrate random combinations of inputs but that a subset receives stereotyped inputs from single projection neurons. This organization maximizes performance of a model output neuron on a stimulus discrimination task. We also report a novel canonical circuit in each mushroom body compartment with previously unidentified connections: reciprocal Kenyon cell to modulatory neuron connections, modulatory neuron to output neuron connections, and a surprisingly high number of recurrent connections between Kenyon cells. Stereotyped connections found between output neurons could enhance the selection of learned behaviours. The complete circuit map of the mushroom body should guide future functional studies of this learning and memory centre.

Dopaminergic neurons (DANs) drive learning across the animal kingdom, but the upstream circuits that regulate their activity and thereby learning remain poorly understood. We provide the first synaptic-resolution connectome of the circuitry upstream of all DANs in a learning center, the mush-room body (MB) of Drosophila larva. We discover afferent sensory pathways and a large population of neurons that provide feedback from MB output neurons and link distinct memory systems (aversive and appetitive). We combine this with functional studies of DANs and their presynaptic partners and with comprehensive circuit modelling. We find that DANs compare convergent feedback from aversive and appetitive systems which enables the computation of integrated predictions that may improve future learning. Computational modelling reveals that the discovered feedback motifs increase model flexibility and performance on learning tasks. Our study provides the most detailed view to date of biological circuit motifs that support associative learning.

An adaptive transition from exploring the environment in search of vital resources to exploiting these resources once the search was successful is important to all animals. Here we study the neuronal circuitry that allows larval Drosophila melanogaster of either sex to negotiate this exploration-exploitation transition. We do so by combining Pavlovian conditioning with high-resolution behavioral tracking, optogenetic manipulation of individually identified neurons, and EM data-based analyses of synaptic organization. We find that optogenetic activation of the dopaminergic neuron DAN-i1 can both establish memory during training and acutely terminate learned search behavior in a subsequent recall test. Its activation leaves innate behavior unaffected, however. Specifically, DAN-i1 activation can establish associative memories of opposite valence after paired and unpaired training with odor, and its activation during the recall test can terminate the search behavior resulting from either of these memories. Our results further suggest that in its behavioral significance DAN-i1 activation resembles, but does not equal, sugar reward. Dendrogram analyses of all the synaptic connections between DAN-i1 and its two main targets, the Kenyon cells and the mushroom body output neuron MBON-i1, further suggest that the DAN-i1 signals during training and during the recall test could be delivered to the Kenyon cells and to MBON-i1, respectively, within previously unrecognized, locally confined branching structures. This would provide an elegant circuit motif to terminate search on its successful completion. SIGNIFICANCE STATEMENT In the struggle for survival, animals have to explore their environment in search of food. Once food is found, however, it is adaptive to prioritize exploiting it over continuing a search that would now be as pointless as searching for the glasses you are wearing. This exploration-exploitation trade-off is important for animals and humans, as well as for technical search devices. We investigate which of the only 10,000 neurons of a fruit fly larva can tip the balance in this trade-off, and identify a single dopamine neuron called DAN-i1 that can do so. Given the similarities in dopamine neuron function across the animal kingdom, this may reflect a general principle of how search is terminated once it is successful.

Amino acids are important nutrients for animals because they are necessary for protein synthesis in particular during growth, as well as for neurotransmission. However, little is known about how animals use past experience to guide their search for amino-acid-rich food. We reasoned that the larvae of

Modulatory (e.g. dopaminergic) neurons provide “teaching signals” that drive associative learning across the animal kingdom, but the circuits that regulate their activity and compute teaching signals are still poorly understood. We provide the first synaptic-resolution connectome of the circuitry upstream of all modulatory neurons in a brain center for associative learning, the mushroom body (MB) of theDrosophilalarva. We discovered afferent pathways from sensory neurons, as well as an unexpected large population of 61 feedback neuron pairs that provide one- and two-step feedback from MB output neurons. The majority of these feedback pathways link distinct memory systems (e.g. aversive and appetitive). We functionally confirmed some of the structural pathways and found that some modulatory neurons compare inhibitory input from their own compartment and excitatory input from compartments of opposite valence, enabling them to compute integrated common-currency predicted values across aversive and appetitive memory systems. This architecture suggests that the MB functions as an interconnected ensemble during learning and that distinct types of previously formed memories can regulate future learning about a stimulus. We developed a model of the circuit constrained by the connectome and by the functional data which revealed that the newly discovered architectural motifs, namely the multilevel feedback architecture and the extensive cross-compartment connections, increase the computational performance and flexibility on learning tasks. Together our study provides the most detailed view to date of a recurrent brain circuit that computes teaching signals and provides insights into the architectural motifs that support reinforcement learning in a biological system.

The

Adjusting behavior to changed environmental contingencies is critical for survival, and reversal learning provides an experimental handle on such cognitive flexibility. Here, we investigate reversal learning in larval Drosophila. Using odor–taste associations, we establish olfactory reversal learning in the appetitive and the aversive domain, using either fructose as a reward or high-concentration sodium chloride as a punishment, respectively. Reversal learning is demonstrated both in differential and in absolute conditioning, in either valence domain. In differential conditioning, the animals are first trained such that an odor A is paired, for example, with the reward whereas odor B is not (A+/B); this is followed by a second training phase with reversed contingencies (A/B+). In absolute conditioning, odor B is omitted, such that the animals are first trained with paired presentations of A and reward, followed by unpaired training in the second training phase. Our results reveal “true” reversal learning in that the opposite associative effects of both the first and the second training phase are detectable after reversed-contingency training. In what is a surprisingly quick, one-trial contingency adjustment in the Drosophila larva, the present study establishes a simple and genetically easy accessible study case of cognitive flexibility.

Amino acids are important nutrients for animals because they are necessary for protein synthesis in particular during growth, as well as for neurotransmission. However, little is known about how animals use past experience to guide their search for amino-acid-rich food. We reasoned that the larvae of Drosophila melanogaster are suitable for investigating this topic because they are the feeding and growth stages in the life cycle of these holometabolous insects. Specifically, we investigated whether experiencing an odour with a 20-amino-acid mixture as a semi-natural tastant during training establishes odour-tastant associative memories. Across a broad concentration range (0.01-20 mM), such an amino-acid mixture was found to have a rewarding effect, establishing appetitive memory for the odour. To our surprise, however, manipulation of the test conditions revealed that relatively high concentrations of the amino-acid mixture (3.3 mM and higher) in addition establish aversive memory for the odour. Both these oppositely-valenced memories we then characterized in terms of their dependency on the number of training trials, their temporal stability, their modulation through starvation, and the specific changes in locomotion underlying them. Collectively, and in the light of what is known about the neuronal organization of odour-food memory in larval Drosophila, our data suggest that these memories are established in parallel. We discuss the similarity of our results to what has been reported for sodium chloride, and the possible neurogenetic bases for concentration-dependent changes in valence when these tastants are used as reinforcers.

The Sap47 gene of Drosophila melanogaster encodes a highly abundant 47 kDa synaptic vesicle-associated protein. Sap47 null mutants show defects in synaptic plasticity and larval olfactory associative learning but the molecular function of Sap47 at the synapse is unknown. We demonstrate that Sap47 modulates the phosphorylation of another highly abundant conserved presynaptic protein, synapsin. Site-specific phosphorylation of Drosophila synapsin has repeatedly been shown to be important for behavioural plasticity but it was not known where these phospho-synapsin isoforms are localized in the brain. Here, we report the distribution of serine-6-phosphorylated synapsin in the adult brain and show that it is highly enriched in rings of synapses in the ellipsoid body and in large synapses near the lateral triangle. The effects of knockout of Sap47 or synapsin on olfactory associative learning/memory support the hypothesis that both proteins operate in the same molecular pathway. We therefore asked if this might also be true for other aspects of their function. We show that knockout of Sap47 but not synapsin reduces lifespan, whereas knockout of Sap47 and synapsin, either individually or together, affects climbing proficiency, as well as plasticity in circadian rhythms and sleep. Furthermore, electrophysiological assessment of synaptic properties at the larval neuromuscular junction (NMJ) reveals increased spontaneous synaptic vesicle fusion and reduced paired pulse facilitation in Sap47 and synapsin single and double mutants. Our results imply that Sap47 and synapsin cooperate non-uniformly in the control of synaptic properties in different behaviourally relevant neuronal networks of the fruitfly.

Punishment feels bad, but relief upon its termination feels good. As a consequence of such timing-dependent valence reversal, memories of opposite valence can result from associating stimulus A with, for example, the occurrence of punishment (A−) versus punishment termination (−A): A− training results in aversive memory, but −A training in appetitive memory (corresponding effects exist for reward occurrence and termination). Whereas learning through the occurrence of punishment is well studied, much less is known about learning through its termination. Current research investigates how dopaminergic system function contributes to these processes in Drosophila, rats and humans. We argue that dopamine-related psychopathology may entail distortions in learning through punishment termination, and that this may contribute, for example, to non-suicidal self-injury or post-traumatic stress disorder.

Animals of many species are capable of “small data” learning, that is, of learning without repetition. Here we introduce larval Drosophila melanogaster as a relatively simple study case for such one-trial learning. Using odor-food associative conditioning, we first show that a sugar that is both sweet and nutritious (fructose) and sugars that are only sweet (arabinose) or only nutritious (sorbitol) all support appetitive one-trial learning. The same is the case for the optogenetic activation of a subset of dopaminergic neurons innervating the mushroom body, the memory center of the insects. In contrast, no one-trial learning is observed for an amino acid reward (aspartic acid). As regards the aversive domain, one-trial learning is demonstrated for high-concentration sodium chloride, but is not observed for a bitter tastant (quinine). Second, we provide follow-up, parametric analyses of odor-fructose learning. Specifically, we ascertain its dependency on the number and duration of training trials, the requirements for the behavioral expression of one-trial odor-fructose memory, its temporal stability, and the feasibility of one-trial differential conditioning. Our results set the stage for a neurogenetic analysis of one-trial learning and define the requirements for modeling mnemonic processes in the larva.

Inspired from plant medicine, a drug that partially compensates for memory decline in aged flies and mice is identified., Cognitive impairments can be devastating for quality of life, and thus, preventing or counteracting them is of great value. To this end, the present study exploits the potential of the plant Rhodiola rosea and identifies the constituent ferulic acid eicosyl ester [icosyl-(2E)-3-(4-hydroxy-3-methoxyphenyl)-prop-2-enoate (FAE-20)] as a memory enhancer. We show that food supplementation with dried root material from R. rosea dose-dependently improves odor-taste reward associative memory scores in larval Drosophila and prevents the age-related decline of this appetitive memory in adult flies. Task-relevant sensorimotor faculties remain unaltered. From a parallel approach, a list of candidate compounds has been derived, including R. rosea–derived FAE-20. Here, we show that both R. rosea–derived FAE-20 and synthetic FAE-20 are effective as memory enhancers in larval Drosophila. Synthetic FAE-20 also partially compensates for age-related memory decline in adult flies, as well as genetically induced early-onset loss of memory function in young flies. Furthermore, it increases excitability in mouse hippocampal CA1 neurons, leads to more stable context-shock aversive associative memory in young adult (3-month-old) mice, and increases memory scores in old (>2-year-old) mice. Given these effects, and given the utility of R. rosea—the plant from which we discovered FAE-20—as a memory enhancer, these results may hold potential for clinical applications.

Painful events establish opponent memories: cues that precede pain are remembered negatively, whereas cues that follow pain, thus coinciding with relief are recalled positively. How do individual reinforcement-signaling neurons contribute to this “timing-dependent valence-reversal?” We addressed this question using an optogenetic approach in the fruit fly. Two types of fly dopaminergic neuron, each comprising just one paired cell, indeed established learned avoidance of odors that preceded their photostimulation during training, and learned approach to odors that followed the photostimulation. This is in striking parallel to punishment versus relief memories reinforced by a real noxious event. For only one of these neuron types, both effects were strong enough for further analyses. Notably, interfering with dopamine biosynthesis in these neurons partially impaired the punishing effect, but not the relieving after-effect of their photostimulation. We discuss how this finding constraints existing computational models of punishment versus relief memories and introduce a new model, which also incorporates findings from mammals. Furthermore, whether using dopaminergic neuron photostimulation or a real noxious event, more prolonged punishment led to stronger relief. This parametric feature of relief may also apply to other animals and may explain particular aspects of related behavioral dysfunction in humans.

Associating stimuli with positive or negative reinforcement is essential for survival, but a complete wiring diagram of a higherorder circuit supporting associative memory has not been previously available. We reconstructed one such circuit at synaptic resolution, theDrosophilalarval mushroom body, and found that most Kenyon cells integrate random combinations of inputs but a subset receives stereotyped inputs from single projection neurons. This organization maximizes performance of a model output neuron on a stimulus discrimination task. We also report a novel canonical circuit in each mushroom body compartment with previously unidentified connections: reciprocal Kenyon cell to modulatory neuron connections, modulatory neuron to output neuron connections, and a surprisingly high number of recurrent connections between Kenyon cells. Stereotyped connections between output neurons could enhance the selection of learned responses. The complete circuit map of the mushroom body should guide future functional studies of this learning and memory center.

Mapping brain function to brain structure is a fundamental task for neuroscience. For such an endeavour, the Drosophila larva is simple enough to be tractable, yet complex enough to be interesting. It features about 10,000 neurons and is capable of various taxes, kineses and Pavlovian conditioning. All its neurons are currently being mapped into a light-microscopical atlas, and Gal4 strains are being generated to experimentally access neurons one at a time. In addition, an electron microscopic reconstruction of its nervous system seems within reach. Notably, this electron microscope-based connectome is being drafted for a stage 1 larva - because stage 1 larvae are much smaller than stage 3 larvae. However, most behaviour analyses have been performed for stage 3 larvae because their larger size makes them easier to handle and observe. It is therefore warranted to either redo the electron microscopic reconstruction for a stage 3 larva or to survey the behavioural faculties of stage 1 larvae. We provide the latter. In a community-based approach we called the Ol1mpiad, we probed stage 1 Drosophila larvae for free locomotion, feeding, responsiveness to substrate vibration, gentle and nociceptive touch, burrowing, olfactory preference and thermotaxis, light avoidance, gustatory choice of various tastants plus odour-taste associative learning, as well as light/dark-electric shock associative learning. Quantitatively, stage 1 larvae show lower scores in most tasks, arguably because of their smaller size and lower speed. Qualitatively, however, stage 1 larvae perform strikingly similar to stage 3 larvae in almost all cases. These results bolster confidence in mapping brain structure and behaviour across developmental stages. The Gulbenkian/Melbourne group was supported by the Fundação para a Ciência e a Tecnologia (FCT; post-doctoral fellowship to M.J.A.-C.: SFRH/BPD/75993/2011; exploratory grant to M.J.A.-C. and C.K.M.: EXPL/BEX-BID/0497/2013). The Münster group was supported by the Cluster of Excellence Cells in Motion (CiM) and the CiM International Max Planck research school (CiM-IMPRS). The Barcelona group was supported by the Spanish Ministry of Economy and Competitiveness, 'Centro de Excelencia Severo Ochoa 2013-2017' (SEV-2012-0208), the CERCA Programme/Generalitat de Catalunya, the 'la Caixa' International PhD Programme, and the Spanish Ministry of Science and Innovation (BFU2011-26208). The LIN-GoLM group received support from the Wissenschaftsgemeinschaft Gottfried Wilhelm Leibniz, the State of Sachsen-Anhalt, the Center for Behavioral Brain Sciences Magdeburg and the Otto von Guericke Universität Magdeburg, as well as from the Deutsche Forschungsgemeinschaft (CRC 779 Motivated behaviour: B11; GE1091/4-1) and the European Commission (FP7-ICT project Miniature Insect Model for Active Learning MINIMAL). The Janelia group received support from the Howard Hughes Medical Institute. The Fribourg group was supported by a starter grant from the European Research Council (ERC-2012-StG 309832-PhotoNaviNet) and the Swiss National Science Foundation (31003A_169993). The Hamburg group was supported by the Deutsche Forschungsgemeinschaft (SPP 1926, Next generation optogenetics, SO1337/2-1), and the Landesforschungsförderung Hamburg (LFF-FV27). The LIN-MolSysBiol group received support from the Wissenschaftsgemeinschaft Gottfried Wilhelm Leibniz, the State of Sachsen-Anhalt, the Center for Behavioral Brain Sciences Magdeburg and the Deutsche Forschungsgemeinschaft (CRC 779 Motivated behaviour: B15; YA272/2-1). The Bonn group was supported by the Deutsche Forschungsgemeinschaft (PA 787/7-1) and Cluster of Excellence ImmunoSensation. The Konstanz group was supported by Deutsche Forschungsgemeinschaft (TH1584/1-1, TH1584/3-1), the Baden-Württemberg Stiftung and the Zukunftskolleg of the University of Konstanz.

Larval Drosophila offer a study case for behavioral neurogenetics that is simple enough to be experimentally tractable, yet complex enough to be worth the effort. We provide a detailed, hands-on manual for Pavlovian odor-reward learning in these animals. Given the versatility of Drosophila for genetic analyses, combined with the evolutionarily shared genetic heritage with humans, the paradigm has utility not only in behavioral neurogenetics and experimental psychology, but for translational biomedicine as well. Together with the upcoming total synaptic connectome of the Drosophila nervous system and the possibilities of single-cell-specific transgene expression, it offers enticing opportunities for research. Indeed, the paradigm has already been adopted by a number of labs and is robust enough to be used for teaching in classroom settings. This has given rise to a demand for a detailed, hands-on manual directed at newcomers and/or at laboratory novices, and this is what we here provide. The paradigm and the present manual have a unique set of features: (1) The paradigm is cheap, easy, and robust; (2) The manual is detailed enough for newcomers or laboratory novices; (3) It briefly covers the essential scientific context; (4) It includes sheets for scoring, data analysis, and display; (5) It is multilingual: in addition to an English version we provide German, French, Japanese, Spanish and Italian language versions as well. The present manual can thus foster science education at an earlier age and enable research by a broader community than has been the case to date.

Mapping brain function to brain structure is a fundamental task for neuroscience. For such an endeavour, the

New techniques and resources for remote control of neuronal activity at single-cell resolution have led to an explosion of research into the behavioral neurogenetics of the memory trace (engram) in the fruit fly Drosophila . The available data suggest that after odor-punishment training, associative and odor-specific engrams are local to a single layer of processing along the olfactory pathway, the presynaptic output terminals of the mushroom body Kenyon cells. At the same time, engrams are distributed across this layer for different mnemonic aspects of such a training event, and for different kinds of training event. This nature of engrams as being local and distributed can be revealed at due resolution in Drosophila – as can the way learned behavior is organized in an outcome-oriented way.

Amino acids are important nutrients for animals, reflected in conserved internal pathways in vertebrates and invertebrates for monitoring cellular levels of these compounds. In mammals, sensory cells and metabotropic glutamate receptor-related taste receptors that detect environmental sources of amino acids in food are also well-characterised. By contrast, it is unclear how insects perceive this class of molecules through peripheral chemosensory mechanisms. Here we investigate amino acid sensing in Drosophila melanogaster larvae, which feed ravenously to support their rapid growth. We show that larvae display diverse behaviours (attraction, aversion, neutral) towards different amino acids, which depend upon stimulus concentration. Some of these behaviours require IR76b, a member of the variant ionotropic glutamate receptor repertoire of invertebrate chemoreceptors. IR76b is broadly expressed in larval taste neurons, suggesting a role as a co-receptor. We identify a subpopulation of these neurons that displays physiological activation by some, but not all, amino acids, and which mediate suppression of feeding by high concentrations of at least a subset of these compounds. Our data reveal the first elements of a sophisticated neuronal and molecular substrate by which these animals detect and behave towards external sources of amino acids.

Dopaminergic neurons serve multiple functions, including reinforcement processing during associative learning [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12]. It is thus warranted to understand which dopaminergic neurons mediate which function. We study larval Drosophila, in which only approximately 120 of a total of 10,000 neurons are dopaminergic, as judged by the expression of tyrosine hydroxylase (TH), the rate- limiting enzyme of dopamine biosynthesis [ 5 and 13]. Dopaminergic neurons mediating reinforcement in insect olfactory learning target the mushroom bodies, a higher-order “cortical” brain region [ 1, 2, 3, 4, 5, 11, 12, 14 and 15]. We discover four previously undescribed paired neurons, the primary protocerebral anterior medial (pPAM) neurons. These neurons are TH positive and subdivide the medial lobe of the mushroom body into four distinct subunits. These pPAM neurons are acutely necessary for odor-sugar reward learning and require intact TH function in this process. However, they are dispensable for aversive learning and innate behavior toward the odors and sugars employed. Optogenetical activation of pPAM neurons is sufficient as a reward. Thus, the pPAM neurons convey a likely dopaminergic reward signal. In contrast, DL1 cluster neurons convey a corresponding punishment signal [5], suggesting a cellular division of labor to convey dopaminergic reward and punishment signals. On the level of individually identified neurons, this uncovers an organizational principle shared with adult Drosophila and mammals [ 1, 2, 3, 4, 7, 9 and 10] (but see [6]). The numerical simplicity and connectomic tractability of the larval nervous system [ 16, 17, 18 and 19] now offers a prospect for studying circuit principles of dopamine function at unprecedented resolution.

Synapsin is an evolutionarily conserved presynaptic phosphoprotein. It is encoded by only one gene in the Drosophila genome and is expressed throughout the nervous system. It regulates the balance between reserve and releasable vesicles, is required to maintain transmission upon heavy demand, and is essential for proper memory function at the behavioral level. Task-relevant sensorimotor functions, however, remain intact in the absence of Synapsin. Using an odor–sugar reward associative learning paradigm in larval Drosophila, we show that memory scores in mutants lacking Synapsin (syn97) are lower than in wild-type animals only when more salient, higher concentrations of odor or of the sugar reward are used. Furthermore, we show that Synapsin is selectively required for larval short-term memory. Thus, without Synapsin Drosophila larvae can learn and remember, but Synapsin is required to form memories that match in strength to event salience—in particular to a high saliency of odors, of rewards, or the salient recency of an event. We further show that the residual memory scores upon a lack of Synapsin are not further decreased by an additional lack of the Sap47 protein. In combination with mass spectrometry data showing an up-regulated phosphorylation of Synapsin in the larval nervous system upon a lack of Sap47, this is suggestive of a functional interdependence of Synapsin and Sap47.

How do animals adaptively integrate innate with learned behavioral tendencies? We tackle this question using chemotaxis as a paradigm. Chemotaxis in the Drosophila larva largely results from a sequence of runs and oriented turns. Thus, the larvae minimally need to determine (i) how fast to run, (ii) when to initiate a turn, and (iii) where to direct a turn. We first report how odor-source intensities modulate these decisions to bring about higher levels of chemotactic performance for higher odor-source intensities during innate chemotaxis. We then examine whether the same modulations are responsible for alterations of chemotactic performance by learned odor "valence" (understood throughout as level of attractiveness). We find that run speed (i) is neither modulated by the innate nor by the learned valence of an odor. Turn rate (ii), however, is modulated by both: the higher the innate or learned valence of the odor, the less often larvae turn whenever heading toward the odor source, and the more often they turn when heading away. Likewise, turning direction (iii) is modulated concordantly by innate and learned valence: turning is biased more strongly toward the odor source when either innate or learned valence is high. Using numerical simulations, we show that a modulation of both turn rate and of turning direction is sufficient to account for the empirically found differences in preference scores across experimental conditions. Our results suggest that innate and learned valence organize adaptive olfactory search behavior by their summed effects on turn rate and turning direction, but not on run speed. This work should aid studies into the neural mechanisms by which memory impacts specific aspects of behavior. We received project support by the European Commission (FP7-ICT project Miniature Insect Model for Active Learning [MINIMAL]). M.L. and S.F.R. acknowledge further funding from the Spanish Ministry of Science and Innovation (MICINN, BFU2011-483 26208) and the EMBL/CRG Systems Biology Program. A.D. acknowledges support via the grants EP/F500385/1 and BB/F529254/1 for the University of Edinburgh School of Informatics Doctoral Training Centre in Neuroinformatics and Computational Neuroscience, the UK Engineering and Physical Sciences Research Council (EPSRC), UK Biotechnology and Biological Sciences Research Council (BBSRC), and the UK Medical Research Council (MRC). B.G. received additional support from the Deutsche Forschungsgemeinschaft (DFG) (SFB 779 Motivated behavior), and the Bundesministerium fu¨r Bildung und Forschung (BMBF) (Bernstein Network Insect inspired robotics).

Adverse life events can induce two kinds of memory with opposite valence, dependent on timing: “negative” memories for stimuli preceding them and “positive” memories for stimuli experienced at the moment of “relief.” Such punishment memory and relief memory are found in insects, rats, and man. For example, fruit flies (Drosophila melanogaster) avoid an odor after odor-shock training (“forward conditioning” of the odor), whereas after shock-odor training (“backward conditioning” of the odor) they approach it. Do these timing-dependent associative processes share molecular determinants? We focus on the role of Synapsin, a conserved presynaptic phosphoprotein regulating the balance between the reserve pool and the readily releasable pool of synaptic vesicles. We find that a lack of Synapsin leaves task-relevant sensory and motor faculties unaffected. In contrast, both punishment memory and relief memory scores are reduced. These defects reflect a true lessening of associative memory strength, as distortions in nonassociative processing (e.g., susceptibility to handling, adaptation, habituation, sensitization), discrimination ability, and changes in the time course of coincidence detection can be ruled out as alternative explanations. Reductions in punishment- and relief-memory strength are also observed upon an RNAi-mediated knock-down of Synapsin, and are rescued both by acutely restoring Synapsin and by locally restoring it in the mushroom bodies of mutant flies. Thus, both punishment memory and relief memory require the Synapsin protein and in this sense share genetic and molecular determinants. We note that corresponding molecular commonalities between punishment memory and relief memory in humans would constrain pharmacological attempts to selectively interfere with excessive associative punishment memories, e.g., after traumatic experiences.