IR76b-expressing neurons in Drosophila melanogaster are necessary for associative reward learning of an amino acid mixture.

Learning where to find nutrients while at the same time avoiding toxic food is essential for survival of any animal. Using Drosophila melanogaster larvae as a study case, we investigate the role of gustatory sensory neurons expressing IR76b for associative learning of amino acids, the building blocks of proteins. We found surprising complexity in the neuronal underpinnings of sensing amino acids, and a functional division of sensory neurons. We found that the IR76b receptor is dispensable for amino acid learning, whereas the neurons expressing IR76b are specifically required for the rewarding but not the punishing effect of amino acids. This unexpected dissociation in neuronal processing of amino acids for different behavioural functions provides a study case for functional divisions of labour in gustatory systems.

Associative learning in larval and adult Drosophila is impaired by the dopamine-synthesis inhibitor 3-Iodo-L-tyrosine.

Across the animal kingdom, dopamine plays a crucial role in conferring reinforcement signals that teach animals about the causal structure of the world. In the fruit fly Drosophila melanogaster, dopaminergic reinforcement has largely been studied using genetics, whereas pharmacological approaches have received less attention. Here, we apply the dopamine-synthesis inhibitor 3-Iodo-L-tyrosine (3IY), which causes acute systemic inhibition of dopamine signaling, and investigate its effects on Pavlovian conditioning. We find that 3IY feeding impairs sugar-reward learning in larvae while leaving task-relevant behavioral faculties intact, and that additional feeding of a precursor of dopamine (L-3,4-dihydroxyphenylalanine, L-DOPA), rescues this impairment. Concerning a different developmental stage and for the aversive valence domain. Moreover, we demonstrate that punishment learning by activating the dopaminergic neuron PPL1-γ1pedc in adult flies is also impaired by 3IY feeding, and can likewise be rescued by L-DOPA. Our findings exemplify the advantages of using a pharmacological approach in combination with the genetic techniques available in D. melanogaster to manipulate neuronal and behavioral function.

An amino-acid mixture can be both rewarding and punishing to larval Drosophilamelanogaster.

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 mmol l-1), 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 mmol l-1 and higher) in addition establish aversive memory for the odour. We then characterized both of these oppositely valenced memories 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 D. melanogaster, 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.

Neuronal processing of amino acids in Drosophila: from taste sensing to behavioural regulation.

Finding and feeding on appropriate food are crucial for all animals. Carbohydrates and amino acids are both essential nutrients, albeit with distinct roles: the former are the main energy source whereas the latter are the building blocks of proteins and are used as neurotransmitters. Despite their crucial role, neither the sensing nor the neuronal processing of amino acids is well understood. Studies in Drosophila melanogaster have only recently gained momentum in shedding new light on the molecular and neuronal mechanisms of peripheral and internal amino acid sensing, as well as the organization of amino acid feeding behaviour. Furthermore, amino acids have been shown to act as rewards in associative learning. Focusing on recent studies in Drosophila, we summarize what is known so far about the perception of, and the behavioural responses to, amino acids in insects, and try to identify key questions for future research.

The Ol1mpiad: concordance of behavioural faculties of stage 1 and stage 3 Drosophila larvae.

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.

Pavlovian Conditioning of Larval Drosophila: An Illustrated, Multilingual, Hands-On Manual for Odor-Taste Associative Learning in Maggots.

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: The paradigm is cheap, easy, and robust;The manual is detailed enough for newcomers or laboratory novices;It briefly covers the essential scientific context;It includes sheets for scoring, data analysis, and display;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.

Preference for and learning of amino acids in larval Drosophila.

Relative to other nutrients, less is known about how animals sense amino acids and how behaviour is organized accordingly. This is a significant gap in our knowledge because amino acids are required for protein synthesis - and hence for life as we know it. Choosing Drosophila larvae as a case study, we provide the first systematic analysis of both the preference behaviour for, and the learning of, all 20 canonical amino acids in Drosophila We report that preference for individual amino acids differs according to the kind of amino acid, both in first-instar and in third-instar larvae. Our data suggest that this preference profile changes across larval instars, and that starvation during the third instar also alters this profile. Only aspartic acid turns out to be robustly attractive across all our experiments. The essentiality of amino acids does not appear to be a determinant of preference. Interestingly, although amino acids thus differ in their innate attractiveness, we find that all amino acids are equally rewarding. Similar discrepancies between innate attractiveness and reinforcing effect have previously been reported for other tastants, including sugars, bitter substances and salt. The present analyses will facilitate the ongoing search for the receptors, sensory neurons, and internal, homeostatic amino acid sensors in Drosophila.

Octopamine and Tyramine Contribute Separately to the Counter-Regulatory Response to Sugar Deficit in Drosophila.

All animals constantly negotiate external with internal demands before and during action selection. Energy homeostasis is a major internal factor biasing action selection. For instance, in addition to physiologically regulating carbohydrate mobilization, starvation-induced sugar shortage also biases action selection toward food-seeking and food consumption behaviors (the counter-regulatory response). Biogenic amines are often involved when such widespread behavioral biases need to be orchestrated. In mammals, norepinephrine (noradrenalin) is involved in the counterregulatory response to starvation-induced drops in glucose levels. The invertebrate homolog of noradrenalin, octopamine (OA) and its precursor tyramine (TA) are neuromodulators operating in many different neuronal and physiological processes. Tyrosine-ß-hydroxylase (tßh) mutants are unable to convert TA into OA. We hypothesized that tßh mutant flies may be aberrant in some or all of the counter-regulatory responses to starvation and that techniques restoring gene function or amine signaling may elucidate potential mechanisms and sites of action. Corroborating our hypothesis, starved mutants show a reduced sugar response and their hemolymph sugar concentration is elevated compared to control flies. When starved, they survive longer. Temporally controlled rescue experiments revealed an action of the OA/TA-system during the sugar response, while spatially controlled rescue experiments suggest actions also outside of the nervous system. Additionally, the analysis of two OA- and four TA-receptor mutants suggests an involvement of both receptor types in the animals' physiological and neuronal response to starvation. These results complement the investigations in Apis mellifera described in our companion paper (Buckemüller et al., 2017).

Genetic variation in food choice behaviour of amino acid-deprived Drosophila.

To understand homeostatic regulation in insects, we need to understand the mechanisms by which they respond to external stimuli to maintain the internal milieu. Our previous study showed that Drosophila melanogaster exhibit specific amino acid preferences. Here, we used the D.melanogaster Genetic Reference Panel (DGRP), which is comprised of multiple inbred lines derived from a natural population, to examine how amino acid preference changes depending on the internal nutritional state in different lines. We performed a two-choice preference test and observed genetic variations in the response to amino acid deprivation. For example, a high-responding line showed an enhanced preference for amino acids even after only 1day of deprivation and responded to a fairly low concentration of amino acids. Conversely, a low-responding line showed no increased preference for amino acids after deprivation. We compared the gene expression profiles between selected high- and the low-responding lines and performed SNP analyses. We found several groups of genes putatively involved in altering amino acid preference. These results will contribute to future studies designed to explore how the genetic architecture of an organism evolves to adapt to different nutritional environments.

Taste preference for amino acids is dependent on internal nutritional state in Drosophila melanogaster.

Like mammals, insects need to ingest proteins from foods because they cannot synthesise several amino acids. Amino acids are also essential nutrients for Drosophila melanogaster, especially for female egg production, but how flies detect amino acids and how the feeding response to amino acids is regulated are unknown. In this study, the two-choice preference test, the proboscis extension reflex test and a CAFE assay were performed to explore the ability of D. melanogaster to detect and discriminate amino acids. To determine whether D. melanogaster change their feeding preference to amino acids after being deprived of them, as previously reported in the locust, two groups of flies raised on the usual medium or on glucose medium were compared. Amino-acid-deprived flies demonstrated enhanced preference to an amino acid mixture and to several amino acids. These flies ingested amino acids even when they were replete with glucose. The proboscis extension reflex to particular amino acids was induced only in amino-acid-deprived flies. Our findings indicate that the sensitivity of labellar taste cells to amino acids may change when flies are deficient in amino acid supply, and also reveal that the detection pathways for individual amino acids may differ. We suggest the existence of an amino acid receptor and a monitoring system regulating the feeding responses to amino acids.