A population of neurons that produce hugin and express the diuretic hormone 44 receptor gene projects to the corpora allata in Drosophila melanogaster.

The corpora allata (CA) are essential endocrine organs that biosynthesize and secrete the sesquiterpenoid hormone, namely juvenile hormone (JH), to regulate a wide variety of developmental and physiological events in insects. CA are directly innervated with neurons in many insect species, implying the innervations to be important for regulating JH biosynthesis. Although this is also true for the model organism Drosophila melanogaster, neurotransmitters produced in the CA-projecting neurons are yet to be identified. In this study on D. melanogaster, we aimed to demonstrate that a subset of neurons producing the neuropeptide hugin, the invertebrate counterpart of the vertebrate neuromedin U, directly projects to the adult CA. A synaptic vesicle marker in the hugin neurons was observed at their axon termini located on the CA, which were immunolabeled with a newly-generated antibody to the JH biosynthesis enzyme JH acid O-methyltransferase. We also found the CA-projecting hugin neurons to likely express a gene encoding the specific receptor for diuretic hormone 44 (Dh44). Moreover, our data suggest that the CA-projecting hugin neurons have synaptic connections with the upstream neurons producing Dh44. Unexpectedly, the inhibition of CA-projecting hugin neurons did not significantly alter the expression levels of the JH-inducible gene Krüppel-homolog 1, which implies that the CA-projecting neurons are not involved in JH biosynthesis but rather in other known biological processes. This is the first study to identify a specific neurotransmitter of the CA-projecting neurons in D. melanogaster, and to anatomically characterize a neuronal pathway of the CA-projecting neurons and their upstream neurons.

Selection of motor programs for suppressing food intake and inducing locomotion in the Drosophila brain.

Central mechanisms by which specific motor programs are selected to achieve meaningful behaviors are not well understood. Using electrophysiological recordings from pharyngeal nerves upon central activation of neurotransmitter-expressing cells, we show that distinct neuronal ensembles can regulate different feeding motor programs. In behavioral and electrophysiological experiments, activation of 20 neurons in the brain expressing the neuropeptide hugin, a homolog of mammalian neuromedin U, simultaneously suppressed the motor program for food intake while inducing the motor program for locomotion. Decreasing hugin neuropeptide levels in the neurons by RNAi prevented this action. Reducing the level of hugin neuronal activity alone did not have any effect on feeding or locomotion motor programs. Furthermore, use of promoter-specific constructs that labeled subsets of hugin neurons demonstrated that initiation of locomotion can be separated from modulation of its motor pattern. These results provide insights into a neural mechanism of how opposing motor programs can be selected in order to coordinate feeding and locomotive behaviors.

Pathogen-induced food evasion behavior in Drosophila larvae.

Recognizing a deadly pathogen and generating an appropriate immune reaction is essential for any organism to survive in its natural habitat. Unlike vertebrates and higher primates, invertebrates depend solely on the innate immune system to defend themselves from an attacking pathogen. In this study, we report a behavioral defense strategy observed in Drosophila larvae that helps them escape and limit an otherwise lethal infection. A bacterial infection in the gut is sensed by the larval central nervous system, which generates an alteration in the larva's food preference, leading it to stop feeding and move away from the infectious food source. We have also found that this behavioral response is dependent on the internal nutritive state of the larvae. Using this novel behavioral assay as a read-out, we further identified hugin neuropeptide to be involved in the evasion response and detection of bacterial signals.

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.

Opposing effects of dietary protein and sugar regulate a transcriptional target of Drosophila insulin-like peptide signaling.

Specific neurosecretory cells of the Drosophila brain express insulin-like peptides (dilps), which regulate growth, glucose homeostasis, and aging. Through microarray analysis of flies in which the insulin-producing cells (IPCs) were ablated, we identified a target gene, target of brain insulin (tobi), that encodes an evolutionarily conserved alpha-glucosidase. Flies with lowered tobi levels are viable, whereas tobi overexpression causes severe growth defects and a decrease in body glycogen. Interestingly, tobi expression is increased by dietary protein and decreased by dietary sugar. This pattern is reminiscent of mammalian glucagon secretion, which is increased by protein intake and decreased by sugar intake, suggesting that tobi is regulated by a glucagon analog. tobi expression is also eliminated upon ablation of neuroendocrine cells that produce adipokinetic hormone (AKH), an analog of glucagon. tobi is thus a target of the insulin- and glucagon-like signaling system that responds oppositely to dietary protein and sugar.

Neuroscience: Moving thoughts control insulin release.

Insulin release has mostly been studied in the context of metabolic signals. An electrophysiology approach in Drosophila now reveals regulation of insulin-producing cell activity by neuronal circuits controlling locomotion. Even without actual movement, activating these circuits is sufficient to inhibit neuropeptide release.

A neuropeptidergic circuit gates selective escape behavior of Drosophila larvae.

Animals display selective escape behaviors when faced with environmental threats. Selection of the appropriate response by the underlying neuronal network is key to maximizing chances of survival, yet the underlying network mechanisms are so far not fully understood. Using synapse-level reconstruction of the Drosophila larval network paired with physiological and behavioral readouts, we uncovered a circuit that gates selective escape behavior for noxious light through acute and input-specific neuropeptide action. Sensory neurons required for avoidance of noxious light and escape in response to harsh touch, each converge on discrete domains of neuromodulatory hub neurons. We show that acute release of hub neuron-derived insulin-like peptide 7 (Ilp7) and cognate relaxin family receptor (Lgr4) signaling in downstream neurons are required for noxious light avoidance, but not harsh touch responses. Our work highlights a role for compartmentalized circuit organization and neuropeptide release from regulatory hubs, acting as central circuit elements gating escape responses.

Unveiling the sensory and interneuronal pathways of the neuroendocrine connectome in Drosophila.

Neuroendocrine systems in animals maintain organismal homeostasis and regulate stress response. Although a great deal of work has been done on the neuropeptides and hormones that are released and act on target organs in the periphery, the synaptic inputs onto these neuroendocrine outputs in the brain are less well understood. Here, we use the transmission electron microscopy reconstruction of a whole central nervous system in the Drosophila larva to elucidate the sensory pathways and the interneurons that provide synaptic input to the neurosecretory cells projecting to the endocrine organs. Predicted by network modeling, we also identify a new carbon dioxide-responsive network that acts on a specific set of neurosecretory cells and that includes those expressing corazonin (Crz) and diuretic hormone 44 (Dh44) neuropeptides. Our analysis reveals a neuronal network architecture for combinatorial action based on sensory and interneuronal pathways that converge onto distinct combinations of neuroendocrine outputs.

Making Feeding Decisions in the Drosophila Nervous System.

Feeding is one of the most fundamental activities of animals. Whether an animal will eat or not depends on sensory cues concerning nutrient availability and quality as well as on its growth, hormonal and metabolic state. These diverse signals, which originate from different regions of the body and act on different time scales, must be integrated by the nervous system to enable an appropriate feeding response. Here, we review recent studies in Drosophila melanogaster larvae that aim to elucidate the central circuits that underlie food intake, based on a serial section electron microscopic volume of an entire central nervous system. We focus on the comprehensive mapping of the synaptic connections between the sensory inputs and motor outputs of the larval feeding system. The central feeding circuit can be organized into a series of parallel pathways that connect a given set of input and output neurons. A dominant circuit motif is that of a monosynaptic sensory-motor connection upon which a series of polysynaptic paths are superimposed. The interneurons of the different parallel paths receive slightly different sets of sensory inputs, which enable flexibility in the selection of feeding motor outputs.

The Corazonin-PTTH Neuronal Axis Controls Systemic Body Growth by Regulating Basal Ecdysteroid Biosynthesis in Drosophila melanogaster.

Steroid hormones play key roles in development, growth, and reproduction in various animal phyla [1]. The insect steroid hormone, ecdysteroid, coordinates growth and maturation, represented by molting and metamorphosis [2]. In Drosophila melanogaster, the prothoracicotropic hormone (PTTH)-producing neurons stimulate peak levels of ecdysteroid biosynthesis for maturation [3]. Additionally, recent studies on PTTH signaling indicated that basal levels of ecdysteroid negatively affect systemic growth prior to maturation [4-8]. However, it remains unclear how PTTH signaling is regulated for basal ecdysteroid biosynthesis. Here, we report that Corazonin (Crz)-producing neurons regulate basal ecdysteroid biosynthesis by affecting PTTH neurons. Crz belongs to gonadotropin-releasing hormone (GnRH) superfamily, implying an analogous role in growth and maturation [9]. Inhibition of Crz neuronal activity increased pupal size, whereas it hardly affected pupariation timing. This phenotype resulted from enhanced growth rate and a delay in ecdysteroid elevation during the mid-third instar larval (L3) stage. Interestingly, Crz receptor (CrzR) expression in PTTH neurons was higher during the mid- than the late-L3 stage. Silencing of CrzR in PTTH neurons increased pupal size, phenocopying the inhibition of Crz neuronal activity. When Crz neurons were optogenetically activated, a strong calcium response was observed in PTTH neurons during the mid-L3, but not the late-L3, stage. Furthermore, we found that octopamine neurons contact Crz neurons in the subesophageal zone (SEZ), transmitting signals for systemic growth. Together, our results suggest that the Crz-PTTH neuronal axis modulates ecdysteroid biosynthesis in response to octopamine, uncovering a regulatory neuroendocrine system in the developmental transition from growth to maturation.

Candidate gustatory interneurons modulating feeding behavior in the Drosophila brain.

Feeding is a fundamental activity of all animals that can be regulated by internal energy status or external sensory signals. We have characterized a zinc finger transcription factor, klumpfuss (klu), which is required for food intake in Drosophila larvae. Microarray analysis indicates that expression of the neuropeptide gene hugin (hug) in the brain is altered in klu mutants and that hug itself is regulated by food signals. Neuroanatomical analysis demonstrates that hug-expressing neurons project axons to the pharyngeal muscles, to the central neuroendocrine organ, and to the higher brain centers, whereas hug dendrites are innervated by external gustatory receptor-expressing neurons, as well as by internal pharyngeal chemosensory organs. The use of tetanus toxin to block synaptic transmission of hug neurons results in alteration of food intake initiation, which is dependent on previous nutrient condition. Our results provide evidence that hug neurons function within a neural circuit that modulates taste-mediated feeding behavior.

Serotonergic network in the subesophageal zone modulates the motor pattern for food intake in Drosophila.

The functional organization of central motor circuits underlying feeding behaviors is not well understood. We have combined electrophysiological and genetic approaches to investigate the regulatory networks upstream of the motor program underlying food intake in the Drosophila larval central nervous system. We discovered that the serotonergic network of the CNS is able to set the motor rhythm frequency of pharyngeal pumping. Pharmacological experiments verified that modulation of the feeding motor pattern is based on the release of serotonin. Classical lesion and laser based cell ablation indicated that the serotonergic neurons in the subesophageal zone represent a redundant network for motor control of larval food intake.

Convergence of monosynaptic and polysynaptic sensory paths onto common motor outputs in a Drosophila feeding connectome.

We reconstructed, from a whole CNS EM volume, the synaptic map of input and output neurons that underlie food intake behavior of Drosophila larvae. Input neurons originate from enteric, pharyngeal and external sensory organs and converge onto seven distinct sensory synaptic compartments within the CNS. Output neurons consist of feeding motor, serotonergic modulatory and neuroendocrine neurons. Monosynaptic connections from a set of sensory synaptic compartments cover the motor, modulatory and neuroendocrine targets in overlapping domains. Polysynaptic routes are superimposed on top of monosynaptic connections, resulting in divergent sensory paths that converge on common outputs. A completely different set of sensory compartments is connected to the mushroom body calyx. The mushroom body output neurons are connected to interneurons that directly target the feeding output neurons. Our results illustrate a circuit architecture in which monosynaptic and multisynaptic connections from sensory inputs traverse onto output neurons via a series of converging paths.

Genetic dissection of neural circuit anatomy underlying feeding behavior in Drosophila: distinct classes of hugin-expressing neurons.

The hugin gene of Drosophila encodes a neuropeptide with homology to mammalian neuromedin U. The hugin-expressing neurons are localized exclusively to the subesophageal ganglion of the central nervous system and modulate feeding behavior in response to nutrient signals. These neurons send neurites to the protocerebrum, the ventral nerve cord, the ring gland, and the pharynx and may interact with the gustatory sense organs. In this study, we have investigated the morphology of the hugin neurons at a single-cell level by using clonal analysis. We show that single cells project to only one of the four major targets. In addition, the neurites of the different hugin cells overlap in a specific brain region lateral to the foramen of the esophagus, which could be a new site of neuropeptide release for feeding regulation. Our study reveals novel complexity in the morphology of individual hugin neurons, which has functional implication for how they coordinate feeding behavior and growth.

Amino acids, taste circuits, and feeding behavior in Drosophila: towards understanding the psychology of feeding in flies and man.

Feeding can be regulated by a variety of external sensory stimuli such as olfaction and gustation, as well as by systemic internal signals of feeding status and metabolic needs. Faced with a major health epidemic in eating-related conditions, such as obesity and diabetes, there is an ever increasing need to dissect and understand the complex regulatory network underlying the multiple aspects of feeding behavior. In this minireview, we highlight the use of Drosophila in studying the neural circuits that control the feeding behavior in response to external and internal signals. In particular, we outline the work on the neuroanatomical and functional characterization of the newly identified hugin neuronal circuit. We focus on the pivotal role of the central nervous system in integrating external and internal feeding-relevant information, thus enabling the organism to make one of the most basic decisions - to eat or not to eat.

Enzyme-free interrogation of RNA sites via primers and oligonucleotides 3'-linked to gold surfaces.

The synthesis of a phosphoramidite is described that was used for the preparation of oligonucleotides with a 3'-terminal thiol, linked to the DNA via a SAM-forming undecyl chain and a nonadsorptive tetraethylene glycol unit. A gold surface featuring oligonucleotide probes allowed for label-free in situ mass spectrometric determination of a nucleotide in subpicomole quantities of an RNA transcript.

Purine and folate metabolism as a potential target of sex-specific nutrient allocation in Drosophila and its implication for lifespan-reproduction tradeoff.

The reallocation of metabolic resources is important for survival during periods of limited nutrient intake. This has an influence on diverse physiological processes, including reproduction, repair, and aging. One important aspect of resource allocation is the difference between males and females in response to nutrient stress. We identified several groups of genes that are regulated in a sex-biased manner under complete or protein starvation. These range from expected differences in genes involved in reproductive physiology to those involved in amino acid utilization, sensory perception, immune response, and growth control. A striking difference was observed in purine and the tightly interconnected folate metabolism upon protein starvation. From these results, we conclude that the purine and folate metabolic pathway is a major point of transcriptional regulation during resource allocation and may have relevance for understanding the physiological basis for the observed tradeoff between reproduction and longevity.

Neuroscience: Hunger Pangs in the Fly Brain.

Which neurons in the brain become engaged when the body is deprived of food? A new study addresses this question using the vinegar fly Drosophila melanogaster, examining a group of neurons in the brain that show alterations in neural activity when flies are satiated or starved.

Central relay of bitter taste to the protocerebrum by peptidergic interneurons in the Drosophila brain.

Bitter is a taste modality associated with toxic substances evoking aversive behaviour in most animals, and the valence of different taste modalities is conserved between mammals and Drosophila. Despite knowledge gathered in the past on the peripheral perception of taste, little is known about the identity of taste interneurons in the brain. Here we show that hugin neuropeptide-containing neurons in the Drosophila larval brain are necessary for avoidance behaviour to caffeine, and when activated, result in cessation of feeding and mediates a bitter taste signal within the brain. Hugin neuropeptide-containing neurons project to the neurosecretory region of the protocerebrum and functional imaging demonstrates that these neurons are activated by bitter stimuli and by activation of bitter sensory receptor neurons. We propose that hugin neurons projecting to the protocerebrum act as gustatory interneurons relaying bitter taste information to higher brain centres in Drosophila larvae.

Synaptic transmission parallels neuromodulation in a central food-intake circuit.

NeuromedinU is a potent regulator of food intake and activity in mammals. In Drosophila, neurons producing the homologous neuropeptide hugin regulate feeding and locomotion in a similar manner. Here, we use EM-based reconstruction to generate the entire connectome of hugin-producing neurons in the Drosophila larval CNS. We demonstrate that hugin neurons use synaptic transmission in addition to peptidergic neuromodulation and identify acetylcholine as a key transmitter. Hugin neuropeptide and acetylcholine are both necessary for the regulatory effect on feeding. We further show that subtypes of hugin neurons connect chemosensory to endocrine system by combinations of synaptic and peptide-receptor connections. Targets include endocrine neurons producing DH44, a CRH-like peptide, and insulin-like peptides. Homologs of these peptides are likewise downstream of neuromedinU, revealing striking parallels in flies and mammals. We propose that hugin neurons are part of an ancient physiological control system that has been conserved at functional and molecular level.