Hungry flies tune to vinegar.

Many molecular signals that represent hunger and satiety in the body have been identified, but relatively little is known about how these factors alter the nervous system to change behavior. Root et al. (2011) report that hunger modulates the sensitivity of specific olfactory sensory neurons in Drosophila and facilitates odor-search behavior.

Layered reward signalling through octopamine and dopamine in Drosophila.

Dopamine is synonymous with reward and motivation in mammals. However, only recently has dopamine been linked to motivated behaviour and rewarding reinforcement in fruitflies. Instead, octopamine has historically been considered to be the signal for reward in insects. Here we show, using temporal control of neural function in Drosophila, that only short-term appetitive memory is reinforced by octopamine. Moreover, octopamine-dependent memory formation requires signalling through dopamine neurons. Part of the octopamine signal requires the α-adrenergic-like OAMB receptor in an identified subset of mushroom-body-targeted dopamine neurons. Octopamine triggers an increase in intracellular calcium in these dopamine neurons, and their direct activation can substitute for sugar to form appetitive memory, even in flies lacking octopamine. Analysis of the ß-adrenergic-like OCTß2R receptor reveals that octopamine-dependent reinforcement also requires an interaction with dopamine neurons that control appetitive motivation. These data indicate that sweet taste engages a distributed octopamine signal that reinforces memory through discrete subsets of mushroom-body-targeted dopamine neurons. In addition, they reconcile previous findings with octopamine and dopamine and suggest that reinforcement systems in flies are more similar to mammals than previously thought.

Novel antennal lobe substructures revealed in the small hive beetle Aethina tumida.

The small hive beetle, Aethina tumida, is an emerging pest of social bee colonies. A. tumida shows a specialized life style for which olfaction seems to play a crucial role. To better understand the olfactory system of the beetle, we used immunohistochemistry and 3-D reconstruction to analyze brain structures, especially the paired antennal lobes (AL), which represent the first integration centers for odor information in the insect brain. The basic neuroarchitecture of the A. tumida brain compares well to the typical beetle and insect brain. In comparison to other insects, the AL are relatively large in relationship to other brain areas, suggesting that olfaction is of major importance for the beetle. The AL of both sexes contain about 70 olfactory glomeruli with no obvious size differences of the glomeruli between sexes. Similar to all other insects including beetles, immunostaining with an antiserum against serotonin revealed a large cell that projects from one AL to the contralateral AL to densely innervate all glomeruli. Immunostaining with an antiserum against tachykinin-related peptides (TKRP) revealed hitherto unknown structures in the AL. Small TKRP-immunoreactive spherical substructures are in both sexes evenly distributed within all glomeruli. The source for these immunoreactive islets is very likely a group of about 80 local AL interneurons. We offer two hypotheses on the function of such structures.

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.

Peptidomics-based phylogeny and biogeography of Mantophasmatodea (Hexapoda).

The insect order Mantophasmatodea was described in 2002. Prior to that time, several generations of entomologists had assumed that all major insect taxa were known; thus, its description was a sensation for zoologists. Since then, a surprising abundance and species diversity of this taxon have been found, particularly in the winter rainfall region of South Africa. To learn more about the evolutionary lineages, speciation, and biogeography of Mantophasmatodea, we applied an unusual peptidomics approach. We collected specimens of almost all known and novel taxa of these insects, developed methods for immediate sample preparation in the field, introduced peptide mass fingerprints for the unambiguous identification of taxa, and subsequently analyzed the most extensive data set on peptide hormones ever compiled for insect taxa. To account for intraspecific variation, we analyzed several individuals per putative species. Increased taxon sampling was preferred over a further increase in the number of characters to optimize the accuracy of phylogenetic analyses. The large data set made it possible to test the validity of using neuropeptide sequences, which coevolve with their respective receptors, to analyze phylogenetic relationships among closely related taxa. Altogether, the data from 71 populations of Mantophasmatodea were sufficient to clearly separate the major clades of Mantophasmatodea, including previously undescribed taxa such as Pachyphasma, Striatophasma, and Austrophasmatidae gen. et sp. nov. "RV." The data confirm the monophyly of Austrophasmatidae and show a relatively recent and extensive radiation in the winter rainfall region of South Africa but also suggest that the species-level diversification of Namibian Mantophasma is less marked than previously thought. We discuss the biogeographical and ecological factors that may have resulted in different regional patterns of endemism and species diversity in Mantophasmatodea. The unique development of the neuroendocrine capa-neurons in the ventral nervous system is described as synapomorphy of Mantophasmatodea + Grylloblattodea and is a further argument for a close relationship between these insect taxa.

Olfactory stimuli and moonwalker SEZ neurons can drive backward locomotion in Drosophila.

How different sensory stimuli are collected, processed, and further transformed into a coordinated motor response is a fundamental question in neuroscience. In particular, the internal and external conditions that drive animals to switch to backward walking and the mechanisms by which the nervous system supports such behavior are still unknown. In fruit flies, moonwalker descending neurons (MDNs) are considered command-type neurons for backward locomotion as they receive visual and mechanosensory inputs and transmit motor-related signals to downstream neurons to elicit backward locomotion. Whether other modalities converge onto MDNs, which central brain neurons activate MDNs, and whether other retreat-driving pathways exist is currently unknown. Here, we show that olfactory stimulation can elicit MDN-mediated backward locomotion. Moreover, we identify the moonwalker subesophageal zone neurons (MooSEZs), a pair of bilateral neurons, which can trigger straight and rotational backward locomotion. MooSEZs act via postsynaptic MDNs and via other descending neurons. Although they respond to olfactory input, they are not required for odor-induced backward walking. Thus, this work reveals an important modality input to MDNs, a novel set of neurons presynaptic to MDNs driving backward locomotion and an MDN-independent backward locomotion pathway.

The Panopticon-Assessing the Effect of Starvation on Prolonged Fly Activity and Place Preference.

Animal behaviours are demonstrably governed by sensory stimulation, previous experience and internal states like hunger. With increasing hunger, priorities shift towards foraging and feeding. During foraging, flies are known to employ efficient path integration strategies. However, general long-term activity patterns for both hungry and satiated flies in conditions of foraging remain to be better understood. Similarly, little is known about how permanent contact chemosensory stimulation affects locomotion. To address these questions, we have developed a novel, simplistic fly activity tracking setup-the Panopticon. Using a 3D-printed Petri dish inset, our assay allows recording of walking behaviour, of several flies in parallel, with all arena surfaces covered by a uniform substrate layer. We tested two constellations of providing food: (i) in single patches and (ii) omnipresent within the substrate layer. Fly tracking is done with FIJI, further assessment, analysis and presentation is done with a custom-built MATLAB analysis framework. We find that starvation history leads to a long-lasting reduction in locomotion, as well as a delayed place preference for food patches which seems to be not driven by immediate hunger motivation.

Differential Role for a Defined Lateral Horn Neuron Subset in Naïve Odor Valence in Drosophila.

Value coding of external stimuli in general, and odor valence in particular, is crucial for survival. In flies, odor valence is thought to be coded by two types of neurons: mushroom body output neurons (MBONs) and lateral horn (LH) neurons. MBONs are classified as neurons that promote either attraction or aversion, but not both, and they are dynamically activated by upstream neurons. This dynamic activation updates the valence values. In contrast, LH neurons receive scaled, but non-dynamic, input from their upstream neurons. It remains unclear how such a non-dynamic system generates differential valence values. Recently, PD2a1/b1 LH neurons were demonstrated to promote approach behavior at low odor concentration in starved flies. Here, we demonstrate that at high odor concentrations, these same neurons contribute to avoidance in satiated flies. The contribution of PD2a1/b1 LH neurons to aversion is context dependent. It is diminished in starved flies, although PD2a1/b1 neural activity remains unchanged, and at lower odor concentration. In addition, PD2a1/b1 aversive effect develops over time. Thus, our results indicate that, even though PD2a1/b1 LH neurons transmit hard-wired output, their effect on valence can change. Taken together, we suggest that the valence model described for MBONs does not hold for LH neurons.

Conservation of the function counts: homologous neurons express sequence-related neuropeptides that originate from different genes.

By means of single-cell matrix assisted laser desorption/ionization time-of-flight mass spectrometry, we analysed neuropeptide expression in all FXPRLamide/pheromone biosynthesis activating neuropeptide synthesizing neurons of the adult tobacco hawk moth, Manduca sexta. Mass spectra clearly suggest a completely identical processing of the pheromone biosynthesis activating neuropeptide-precursor in the mandibular, maxillary and labial neuromeres of the subesophageal ganglion. Only in the pban-neurons of the labial neuromere, products of two neuropeptide genes, namely the pban-gene and the capa-gene, were detected. Both of these genes expressed, amongst others, sequence-related neuropeptides (extended WFGPRLamides). We speculate that the expression of the two neuropeptide genes is a plesiomorph character typical of moths. A detailed examination of the neuroanatomy and the peptidome of the (two) pban-neurons in the labial neuromere of moths with homologous neurons of different insects indicates a strong conservation of the function of this neuroendocrine system. In other insects, however, the labial neurons either express products of the fxprl-gene or products of the capa-gene. The processing of the respective genes is reduced to extended WFGPRLamides in each case and yields a unique peptidome in the labial cells. Thus, sequence-related messenger molecules are always produced in these cells and it seems that the respective neurons recruited different neuropeptide genes for this motif.

The prandial process in flies.

Feeding is fundamental to any heterotroph organism; in its role to quell hunger it overrides most other motivational states. But feeding also literally opens the door to harmful risks, especially for a saprophagous animal like Drosophila; ingestion of poisonous substrate can lead to irreversible damage. Thus feeding incorporates a series of steps with several checkpoints to guarantee that the ingestion remains beneficial and provides a balanced diet, or the feeding process is interrupted. Subsequently, we will summarize and describe the feeding process in Drosophila in a comprehensive manner. We propose eleven distinct steps for feeding, grouped into four categories, to address our current knowledge of prandial regulatory mechanisms in Drosophila.

Neuropeptides in modulation of Drosophila behavior: how to get a grip on their pleiotropic actions.

Neuropeptides constitute a large and diverse class of signaling molecules that are produced by many types of neurons, neurosecretory cells, endocrines and other cells. Many neuropeptides display pleiotropic actions either as neuromodulators, co-transmitters or circulating hormones, while some play these roles concurrently. Here, we highlight pleiotropic functions of neuropeptides and different levels of neuropeptide signaling in the brain, from context-dependent orchestrating signaling by higher order neurons, to local executive modulation in specific circuits. Additionally, orchestrating neurons receive peptidergic signals from neurons conveying organismal internal state cues and relay these to executive circuits. We exemplify these levels of signaling with four neuropeptides, SIFamide, short neuropeptide F, allatostatin-A and leucokinin, each with a specific expression pattern and level of complexity in signaling.

Inhibitory muscarinic acetylcholine receptors enhance aversive olfactory learning in adult Drosophila.

Olfactory associative learning in Drosophila is mediated by synaptic plasticity between the Kenyon cells of the mushroom body and their output neurons. Both Kenyon cells and their inputs from projection neurons are cholinergic, yet little is known about the physiological function of muscarinic acetylcholine receptors in learning in adult flies. Here, we show that aversive olfactory learning in adult flies requires type A muscarinic acetylcholine receptors (mAChR-A), particularly in the gamma subtype of Kenyon cells. mAChR-A inhibits odor responses and is localized in Kenyon cell dendrites. Moreover, mAChR-A knockdown impairs the learning-associated depression of odor responses in a mushroom body output neuron. Our results suggest that mAChR-A function in Kenyon cell dendrites is required for synaptic plasticity between Kenyon cells and their output neurons.

Aversive Learning and Appetitive Motivation Toggle Feed-Forward Inhibition in the Drosophila Mushroom Body.

In Drosophila, negatively reinforcing dopaminergic neurons also provide the inhibitory control of satiety over appetitive memory expression. Here we show that aversive learning causes a persistent depression of the conditioned odor drive to two downstream feed-forward inhibitory GABAergic interneurons of the mushroom body, called MVP2, or mushroom body output neuron (MBON)-γ1pedc>α/ß. However, MVP2 neuron output is only essential for expression of short-term aversive memory. Stimulating MVP2 neurons preferentially inhibits the odor-evoked activity of avoidance-directing MBONs and odor-driven avoidance behavior, whereas their inhibition enhances odor avoidance. In contrast, odor-evoked activity of MVP2 neurons is elevated in hungry flies, and their feed-forward inhibition is required for expression of appetitive memory at all times. Moreover, imposing MVP2 activity promotes inappropriate appetitive memory expression in food-satiated flies. Aversive learning and appetitive motivation therefore toggle alternate modes of a common feed-forward inhibitory MVP2 pathway to promote conditioned odor avoidance or approach.

Activity of defined mushroom body output neurons underlies learned olfactory behavior in Drosophila.

During olfactory learning in fruit flies, dopaminergic neurons assign value to odor representations in the mushroom body Kenyon cells. Here we identify a class of downstream glutamatergic mushroom body output neurons (MBONs) called M4/6, or MBON-ß2ß'2a, MBON-ß'2mp, and MBON-γ5ß'2a, whose dendritic fields overlap with dopaminergic neuron projections in the tips of the ß, ß', and γ lobes. This anatomy and their odor tuning suggests that M4/6 neurons pool odor-driven Kenyon cell synaptic outputs. Like that of mushroom body neurons, M4/6 output is required for expression of appetitive and aversive memory performance. Moreover, appetitive and aversive olfactory conditioning bidirectionally alters the relative odor-drive of M4ß' neurons (MBON-ß'2mp). Direct block of M4/6 neurons in naive flies mimics appetitive conditioning, being sufficient to convert odor-driven avoidance into approach, while optogenetically activating these neurons induces avoidance behavior. We therefore propose that drive to the M4/6 neurons reflects odor-directed behavioral choice.

Sweet taste and nutrient value subdivide rewarding dopaminergic neurons in Drosophila.

Dopaminergic neurons provide reward learning signals in mammals and insects [1-4]. Recent work in Drosophila has demonstrated that water-reinforcing dopaminergic neurons are different to those for nutritious sugars [5]. Here, we tested whether the sweet taste and nutrient properties of sugar reinforcement further subdivide the fly reward system. We found that dopaminergic neurons expressing the OAMB octopamine receptor [6] specifically convey the short-term reinforcing effects of sweet taste [4]. These dopaminergic neurons project to the ß'2 and γ4 regions of the mushroom body lobes. In contrast, nutrient-dependent long-term memory requires different dopaminergic neurons that project to the γ5b regions, and it can be artificially reinforced by those projecting to the ß lobe and adjacent α1 region. Surprisingly, whereas artificial implantation and expression of short-term memory occur in satiated flies, formation and expression of artificial long-term memory require flies to be hungry. These studies suggest that short-term and long-term sugar memories have different physiological constraints. They also demonstrate further functional heterogeneity within the rewarding dopaminergic neuron population.

Copper/zinc superoxide dismutase-like immunoreactivity in the metamorphosing brain of the sphinx moth Manduca sexta.

Cu/Zn superoxide dismutase (SOD) is part of the defense mechanism that protects cells from being damaged by reactive oxygen species. During metamorphosis of the nervous system, neurons undergo various fates, which are all coupled to high metabolic activities, such as proliferation, differentiation, pathfinding, and synaptogenesis. We describe the pattern of SOD immunoreactivity of identified neurons and neuron groups in the brain of Manduca sexta from the late larva through metamorphosis into adult. We focused on neurons of the developing antennal lobes, the optic lobes, and the central brain. Our results indicate the transient expression of SOD during phases in which the neurons develop their final adult identities. Our data also suggest that the SOD immunoreactivity may be used as an indicator for the period in which developing neurons form their synapses. We also observed SOD immunoreactivity within nitric oxide-sensitive cells as characterized by immunolabeling against 3'5'-cyclic guanosine monophosphate and soluble guanylyl cyclase, a novel finding in insects.

Hemichannel composition and electrical synaptic transmission: molecular diversity and its implications for electrical rectification.

Unapposed hemichannels (HCs) formed by hexamers of gap junction proteins are now known to be involved in various cellular processes under both physiological and pathological conditions. On the other hand, less is known regarding how differences in the molecular composition of HCs impact electrical synaptic transmission between neurons when they form intercellular heterotypic gap junctions (GJs). Here we review data indicating that molecular differences between apposed HCs at electrical synapses are generally associated with rectification of electrical transmission. Furthermore, this association has been observed at both innexin and connexin (Cx) based electrical synapses. We discuss the possible molecular mechanisms underlying electrical rectification, as well as the potential contribution of intracellular soluble factors to this phenomenon. We conclude that asymmetries in molecular composition and sensitivity to cellular factors of each contributing hemichannel can profoundly influence the transmission of electrical signals, endowing electrical synapses with more complex functional properties.

Neural correlates of water reward in thirsty Drosophila.

Drinking water is innately rewarding to thirsty animals. In addition, the consumed value can be assigned to behavioral actions and predictive sensory cues by associative learning. Here we show that thirst converts water avoidance into water-seeking in naive Drosophila melanogaster. Thirst also permitted flies to learn olfactory cues paired with water reward. Water learning required water taste and <40 water-responsive dopaminergic neurons that innervate a restricted zone of the mushroom body γ lobe. These water learning neurons are different from those that are critical for conveying the reinforcing effects of sugar. Naive water-seeking behavior in thirsty flies did not require water taste but relied on another subset of water-responsive dopaminergic neurons that target the mushroom body ß' lobe. Furthermore, these naive water-approach neurons were not required for learned water-seeking. Our results therefore demonstrate that naive water-seeking, learned water-seeking and water learning use separable neural circuitry in the brain of thirsty flies.