The adhesion G-protein-coupled receptor mayo/CG11318 controls midgut development in Drosophila.

Adhesion G-protein-coupled receptors (aGPCRs) form a large family of cell surface molecules with versatile tasks in organ development. Many aGPCRs still await their functional and pharmacological deorphanization. Here, we characterized the orphan aGPCR CG11318/mayo of Drosophila melanogaster and found it expressed in specific regions of the gastrointestinal canal and anal plates, epithelial specializations that control ion homeostasis. Genetic removal of mayo results in tachycardia, which is caused by hyperkalemia of the larval hemolymph. The hyperkalemic effect can be mimicked by a raise in ambient potassium concentration, while normal potassium levels in mayoKO mutants can be restored by pharmacological inhibition of potassium channels. Intriguingly, hyperkalemia and tachycardia are caused non-cell autonomously through mayo-dependent control of enterocyte proliferation in the larval midgut, which is the primary function of this aGPCR. These findings characterize the ancestral aGPCR Mayo as a homeostatic regulator of gut development.

Developmental emergence of sleep rhythms enables long-term memory in Drosophila.

In adulthood, sleep-wake rhythms are one of the most prominent behaviors under circadian control. However, during early life, sleep is spread across the 24-hour day. The mechanism through which sleep rhythms emerge, and consequent advantage conferred to a juvenile animal, is unknown. In the second-instar Drosophila larvae (L2), like in human infants, sleep is not under circadian control. We identify the precise developmental time point when the clock begins to regulate sleep in Drosophila, leading to emergence of sleep rhythms in early third-instars (L3). At this stage, a cellular connection forms between DN1a clock neurons and arousal-promoting Dh44 neurons, bringing arousal under clock control to drive emergence of circadian sleep. Last, we demonstrate that L3 but not L2 larvae exhibit long-term memory (LTM) of aversive cues and that this LTM depends upon deep sleep generated once sleep rhythms begin. We propose that the developmental emergence of circadian sleep enables more complex cognitive processes, including the onset of enduring memories.

Rewarding Capacity of Optogenetically Activating a Giant GABAergic Central-Brain Interneuron in Larval Drosophila.

Larvae of the fruit fly Drosophila melanogaster are 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 naive 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 STATEMENT The 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 larval Drosophila as 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.

The making of a maggot brain.

The way neurons in the brain rewire in larvae as they turn to adult fruit flies sheds light on how complete metamorphosis was 'invented' over the course of evolution.

Learning and Memory in Drosophila Larvae.

The Drosophila larva has become an attractive model system for studying fundamental questions in neuroscience. Although the focus was initially on topics such as the structure of genes, mechanisms of inheritance, genetic regulation of development, and the function and physiology of ion channels, today it is often on the cellular and molecular principles of naive and learned behavior. Drosophila larvae have developed different mechanisms, often widespread in similar manifestations in the animal kingdom, to orient themselves toward olfactory, gustatory, mechanosensory, thermal, and visual stimuli to coordinate their locomotion appropriately. To adapt to changes in the environment, larvae are able to learn to categorize some of these sensory impressions as "good" or "bad." Depending on their relevance and reliability, the larva learns them and constantly updates these memories. Laboratory experiments allow us to parametrically study and describe many of these processes (e.g., olfactory appetitive and aversive memory or visual appetitive and aversive memory). Combining behavioral tests with various neurogenetic techniques allows us to thermally or optogenetically activate or inhibit individual cells during learning, memory consolidation, and memory retrieval. The molecular and genetic bases of larval learning can be analyzed by using specific mutants. The CRISPR-Cas method has established extensive new directions in this area, in addition to the already wide-ranging traditional approaches, like the GAL4/UAS system. The combination of these genetic methods with the simplicity and cost-effectiveness of the introduced behavioral assay provides a platform for discovering the fundamental mechanisms underlying learning and memory formation in the rather simple larval brain.

The Analysis of Aversive Olfactory-Taste Learning and Memory in Drosophila Larvae.

Drosophila larvae are able to associate an odor stimulus with a temporally overlapping teaching signal encoding reward or punishment. Here, we describe a standardized experimental setup that allows the analysis of larval aversive-odor-taste learning and memory. This is a Pavlovian learning experiment with a single training trial in which larvae are presented with two specific odors in succession, one odor together with salt at a high concentration that is harmful to the larva. In the subsequent test, the trained larvae then show avoidance of the salt-paired odor and spend more time near the unpaired odor. To rule out nonassociative effects (such as naive preferences for odors, exposure, or handling effects), two independent groups of larvae are reciprocally trained. Subsequently, the average of the two individual preference values is determined and quantified as a Performance Index (PI), which assigns a numerical value to the larvae's shown behavioral change.

Synchronous and opponent thermosensors use flexible cross-inhibition to orchestrate thermal homeostasis.

Body temperature homeostasis is essential and reliant upon the integration of outputs from multiple classes of cooling- and warming-responsive cells. The computations that integrate these outputs are not understood. Here, we discover a set of warming cells (WCs) and show that the outputs of these WCs combine with previously described cooling cells (CCs) in a cross-inhibition computation to drive thermal homeostasis in larval Drosophila WCs and CCs detect temperature changes using overlapping combinations of ionotropic receptors: Ir68a, Ir93a, and Ir25a for WCs and Ir21a, Ir93a, and Ir25a for CCs. WCs mediate avoidance to warming while cross-inhibiting avoidance to cooling, and CCs mediate avoidance to cooling while cross-inhibiting avoidance to warming. Ambient temperature-dependent regulation of the strength of WC- and CC-mediated cross-inhibition keeps larvae near their homeostatic set point. Using neurophysiology, quantitative behavioral analysis, and connectomics, we demonstrate how flexible integration between warming and cooling pathways can orchestrate homeostatic thermoregulation.

Ethanol-guided behavior in Drosophila larvae.

Chemosensory signals allow vertebrates and invertebrates not only to orient in its environment toward energy-rich food sources to maintain nutrition but also to avoid unpleasant or even poisonous substrates. Ethanol is a substance found in the natural environment of Drosophila melanogaster. Accordingly, D. melanogaster has evolved specific sensory systems, physiological adaptations, and associated behaviors at its larval and adult stage to perceive and process ethanol. To systematically analyze how D. melanogaster larvae respond to naturally occurring ethanol, we examined ethanol-induced behavior in great detail by reevaluating existing approaches and comparing them with new experiments. Using behavioral assays, we confirm that larvae are attracted to different concentrations of ethanol in their environment. This behavior is controlled by olfactory and other environmental cues. It is independent of previous exposure to ethanol in their food. Moreover, moderate, naturally occurring ethanol concentration of 4% results in increased larval fitness. On the contrary, higher concentrations of 10% and 20% ethanol, which rarely or never appear in nature, increase larval mortality. Finally, ethanol also serves as a positive teaching signal in learning and memory and updates valence associated with simultaneously processed odor information. Since information on how larvae perceive and process ethanol at the genetic and neuronal level is limited, the establishment of standardized assays described here is an important step towards their discovery.

Controlling the behaviour of Drosophila melanogaster via smartphone optogenetics.

Invertebrates such as Drosophila melanogaster have proven to be a valuable model organism for studies of the nervous system. In order to control neuronal activity, optogenetics has evolved as a powerful technique enabling non-invasive stimulation using light. This requires light sources that can deliver patterns of light with high temporal and spatial precision. Currently employed light sources for stimulation of small invertebrates, however, are either limited in spatial resolution or require sophisticated and bulky equipment. In this work, we used smartphone displays for optogenetic control of Drosophila melanogaster. We developed an open-source smartphone app that allows time-dependent display of light patterns and used this to activate and inhibit different neuronal populations in both larvae and adult flies. Characteristic behavioural responses were observed depending on the displayed colour and brightness and in agreement with the activation spectra and light sensitivity of the used channelrhodopsins. By displaying patterns of light, we constrained larval movement and were able to guide larvae on the display. Our method serves as a low-cost high-resolution testbench for optogenetic experiments using small invertebrate species and is particularly appealing to application in neuroscience teaching labs.

Identification of Dopaminergic Neurons That Can Both Establish Associative Memory and Acutely Terminate Its Behavioral Expression.

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.

Recurrent architecture for adaptive regulation of learning in the insect brain.

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 a synaptic-resolution connectome of the circuitry upstream of all DANs in a learning center, the mushroom body of Drosophila larva. We discover afferent sensory pathways and a large population of neurons that provide feedback from mushroom body 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 modeling. 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 modeling 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.

Food restriction reconfigures naïve and learned choice behavior in Drosophila larvae.

In many animals, the establishment and expression of food-related memory is limited by the presence of food and promoted by its absence, implying that this behavior is driven by motivation. In the past, this has already been demonstrated in various insects including honeybees and adult Drosophila. For Drosophila larvae, which are characterized by an immense growth and the resulting need for constant food intake, however, knowledge is rather limited. Accordingly, we have analyzed whether starvation modulates larval memory formation or expression after appetitive classical olfactory conditioning, in which an odor is associated with a sugar reward. We show that odor-sugar memory of starved larvae lasts longer than in fed larvae, although the initial performance is comparable. 80 minutes after odor fructose conditioning, only starved but not fed larvae show a reliable odor-fructose memory. This is likely due to a specific increase in the stability of anesthesia-resistant memory (ARM). Furthermore, we observe that starved larvae, in contrast to fed ones, prefer sugars that offer a nutritional benefit in addition to their sweetness. Taken together our work shows that Drosophila larvae adjust the expression of learned and naïve choice behaviors in the absence of food. These effects are only short-lasting probably due to their lifestyle and their higher internal motivation to feed. In the future, the extensive use of established genetic tools will allow us to identify development-specific differences arising at the neuronal and molecular level.

Solvate ionic liquids based on lithium bis(trifluoromethanesulfonyl)imide-glyme systems: coordination in MD simulations with scaled charges.

Equimolar mixtures of lithium bis(trifluoromethanesulfonyl)imide (Li[NTf2]) with triglyme or tetraglyme (small oligoethers) are regarded as a new class of ionic liquids, the so-called solvate ionic liquids. In these mixtures, the glyme molecules wrap around the lithium ions forming crown-ether like [Li(glyme)1]+ complex cations. New molecular dynamics (MD) simulations suggest that the lithium-glyme coordination is stronger than that predicted in a former MD study [K. Shimizu, et al., Phys. Chem. Chem. Phys., 2015, 17, 22321-22335], whereas lithium-NTf2 connections are weaker. The differences between the present and the previous study arise from different starting conditions. Both studies employed charges scaled by a factor of 0.8. As shown by the comparison of MD simulations with and without reduced charges to experiments, charge scaling is necessary in order to obtain data close to experimental results.

Reversal learning in Drosophila larvae.

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.

MEK inhibitor cobimetinib rescues a dRaf mutant lethal phenotype in Drosophila melanogaster.

Since Drosophila melanogaster has proven to be a useful model system to study phenotypes of oncogenic mutations and to identify new anti-cancer drugs, we generated human BRAFV600E homologous dRaf mutant (dRafA572E ) Drosophila melanogaster strains to use these for characterisation of mutant phenotypes and exploit these phenotypes for drug testing. For mutant gene expression, the GAL4/UAS expression system was used. dRafA572E was expressed tissue-specific in the eye, epidermis, heart, wings, secretory glands and in the whole animal. Expression of dRaf A572E under the control of an eye-specific driver led to semi-lethality and a rough eye phenotype. The vast majority of other tissue-specific and ubiquitous drivers led to a lethal phenotype only. The rough eye phenotype was used to test BRAF inhibitor vemurafenib and MEK1/2 inhibitor cobimetinib. There was no phenotype rescue by this treatment. However, a significant rescue of the lethal phenotype was observed under a gut-specific driver. Here, MEK1/2 inhibitor cobimetinib rescued Drosophila larvae to reach pupal stage in 37% of cases as compared to 1% in control experiments. Taken together, the BRAFV600E homolog dRaf A572E exerts mostly lethal effects in Drosophila. Gut-specific dRaf A572E expression might in future be developed further for drug testing.

Reward signaling in a recurrent circuit of dopaminergic neurons and peptidergic Kenyon cells.

Dopaminergic neurons in the brain of the Drosophila larva play a key role in mediating reward information to the mushroom bodies during appetitive olfactory learning and memory. Using optogenetic activation of Kenyon cells we provide evidence that recurrent signaling exists between Kenyon cells and dopaminergic neurons of the primary protocerebral anterior (pPAM) cluster. Optogenetic activation of Kenyon cells paired with odor stimulation is sufficient to induce appetitive memory. Simultaneous impairment of the dopaminergic pPAM neurons abolishes appetitive memory expression. Thus, we argue that dopaminergic pPAM neurons mediate reward information to the Kenyon cells, and in turn receive feedback from Kenyon cells. We further show that this feedback signaling is dependent on short neuropeptide F, but not on acetylcholine known to be important for odor-shock memories in adult flies. Our data suggest that recurrent signaling routes within the larval mushroom body circuitry may represent a mechanism subserving memory stabilization.

Connectomics: Arrested Development.

Connectomics, the reconstruction of neuronal wiring diagrams via electron microscopy, is bringing us closer to understanding how brains organize behavior. But high-resolution imaging of the brain can do more. A new study now provides insights into how neuronal circuits develop.

Drosophila melanogaster cloak their eggs with pheromones, which prevents cannibalism.

Oviparous animals across many taxa have evolved diverse strategies that deter egg predation, providing valuable tests of how natural selection mitigates direct fitness loss. Communal egg laying in nonsocial species minimizes egg predation. However, in cannibalistic species, this very behavior facilitates egg predation by conspecifics (cannibalism). Similarly, toxins and aposematic signaling that deter egg predators are often inefficient against resistant conspecifics. Egg cannibalism can be adaptive, wherein cannibals may benefit through reduced competition and added nutrition, but since it reduces Darwinian fitness, the evolution of anticannibalistic strategies is rife. However, such strategies are likely to be nontoxic because deploying toxins against related individuals would reduce inclusive fitness. Here, we report how D. melanogaster use specific hydrocarbons to chemically mask their eggs from cannibal larvae. Using an integrative approach combining behavioral, sensory, and mass spectrometry methods, we demonstrate that maternally provisioned pheromone 7,11-heptacosadiene (7,11-HD) in the eggshell's wax layer deters egg cannibalism. Furthermore, we show that 7,11-HD is nontoxic, can mask underlying substrates (for example, yeast) when coated upon them, and its detection requires pickpocket 23 (ppk23) gene function. Finally, using light and electron microscopy, we demonstrate how maternal pheromones leak-proof the egg, consequently concealing it from conspecific larvae. Our data suggest that semiochemicals possibly subserve in deceptive functions across taxa, especially when predators rely on chemical cues to forage, and stimulate further research on deceptive strategies mediated through nonvisual sensory modules. This study thus highlights how integrative approaches can illuminate our understanding on the adaptive significance of deceptive defenses and the mechanisms through which they operate.

Connectomics and function of a memory network: the mushroom body of larval Drosophila.

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.

Maggot Instructor: Semi-Automated Analysis of Learning and Memory in Drosophila Larvae.

For several decades, Drosophila has been widely used as a suitable model organism to study the fundamental processes of associative olfactory learning and memory. More recently, this condition also became true for the Drosophila larva, which has become a focus for learning and memory studies based on a number of technical advances in the field of anatomical, molecular, and neuronal analyses. The ongoing efforts should be mentioned to reconstruct the complete connectome of the larval brain featuring a total of about 10,000 neurons and the development of neurogenic tools that allow individual manipulation of each neuron. By contrast, standardized behavioral assays that are commonly used to analyze learning and memory in Drosophila larvae exhibit no such technical development. Most commonly, a simple assay with Petri dishes and odor containers is used; in this method, the animals must be manually transferred in several steps. The behavioral approach is therefore labor-intensive and limits the capacity to conduct large-scale genetic screenings in small laboratories. To circumvent these limitations, we introduce a training device called the Maggot Instructor. This device allows automatic training up to 10 groups of larvae in parallel. To achieve such goal, we used fully automated, computer-controlled optogenetic activation of single olfactory neurons in combination with the application of electric shocks. We showed that Drosophila larvae trained with the Maggot Instructor establish an odor-specific memory, which is independent of handling and non-associative effects. The Maggot Instructor will allow to investigate the large collections of genetically modified larvae in a short period and with minimal human resources. Therefore, the Maggot Instructor should be able to help extensive behavioral experiments in Drosophila larvae to keep up with the current technical advancements. In the longer term, this condition will lead to a better understanding of how learning and memory are organized at the cellular, synaptic, and molecular levels in Drosophila larvae.