Octopamine in male aggression of Drosophila

BACKGROUND: In mammals and humans, noradrenaline is a key modulator of aggression. Octopamine, a closely related biogenic amine, has been proposed to have a similar function in arthropods. However, the effect of octopamine on aggressive behavior is little understood. RESULTS: An automated video analysis of aggression in male Drosophila has been developed, rendering aggression accessible to high-throughput studies. The software detects the lunge, a conspicuous behavioral act unique to aggression. In lunging, the aggressor rears up on his hind legs and snaps down on his opponent. By using the software to eliminate confounding effects, we now show that aggression is almost abolished in mutant males lacking octopamine. This suppression is independent of whether tyramine, the precursor of octopamine, is increased or also depleted. Restoring octopamine synthesis in the brain either throughout life or in adulthood leads to a partial rescue of aggression. Finally, neuronal silencing of octopaminergic and tyraminergic neurons almost completely abolishes lunges. CONCLUSIONS: Octopamine modulates Drosophila aggression. Genetically depleting the animal of octopamine downregulates lunge frequency without a sizable effect on the lunge motor program. This study provides access to the neuronal circuitry mediating this modulation.     

Roles of octopaminergic and dopaminergic neurons in appetitive and aversive memory recall in an insect

Background  

In insect classical conditioning, octopamine (the invertebrate counterpart of noradrenaline) or dopamine has been suggested to mediate reinforcing properties of appetitive or aversive unconditioned stimulus, respectively. However, the roles of octopaminergic and dopaminergic neurons in memory recall have remained unclear.     

Octopamine modulates activity of neural networks in the honey bee antennal lobe

Neuronal plasticity allows an animal to respond to environmental changes by modulating its response to stimuli. In the honey bee (Apis mellifera), the biogenic amine octopamine plays a crucial role in appetitive odor learning, but little is known about how octopamine affects the brain. We investigated its effect in the antennal lobe, the first olfactory center in the brain, using calcium imaging to record background activity and odor responses before and after octopamine application. We show that octopamine increases background activity in olfactory output neurons, while reducing average calcium levels. Odor responses were modulated both upwards and downwards, with more odor response increases in glomeruli with negative or weak odor responses. Importantly, the octopamine effect was variable across glomeruli, odorants, odorant concentrations and animals, suggesting that the octopaminergic network is shaped by plasticity depending on an individual animal’s history and possibly other factors. Using RNA interference, we show that the octopamine receptor AmOA1 (homolog of the Drosophila OAMB receptor) is involved in the octopamine effect. We propose a network model in which octopamine receptors are plastic in their density and located on a subpopulation of inhibitory neurons in a disinhibitory pathway. This would improve odor-coding of behaviorally relevant, previously experienced odors.     

昆虫非典型嗅觉受体Orco的功能和分子结构研究进展

嗅觉受体是参与昆虫嗅觉识别过程的一类重要蛋白。在昆虫的众多嗅觉受体中, 有一类受体明显不同于其他受体, 被称为Orco。该受体基因在不同昆虫种间高度保守, 且表达广泛。Orco在昆虫嗅觉识别过程中发挥关键作用。采用基因突变或RNAi等技术使Orco基因沉默后, 昆虫会出现严重的嗅觉缺陷, 但Orco本身不与气味配体结合, 它与传统嗅觉受体形成复合体Or-Orco, 促进传统嗅觉受体在神经元树突膜上的定位并维持其稳定性, 提高传统嗅觉受体对气味反应的效率。昆虫嗅觉受体的结构与脊椎动物的G蛋白偶联受体相似, 均有7个跨膜区, 但二者的膜拓扑结构相反, 昆虫嗅觉受体的N末端位于细胞质膜内, C末端在细胞质膜外, Orco与传统嗅觉受体通过保守的C末端区域相互作用形成一种新型的配体门控离子通道--Or-Orco复合体。阐明Orco在昆虫嗅觉识别中的功能机制, 可为开创基于昆虫嗅觉行为干扰的新的害虫防治措施提供基础。     

Neurotoxicity and Mode of Action of N,N-Diethyl-Meta-Toluamide (DEET)

Recent studies suggest that N, N-diethyl-meta-toluamide (DEET) is an acetylcholinesterase inhibitor and that this action may result in neurotoxicity and pose a risk to humans from its use as an insect repellent. We investigated the mode of action of DEET neurotoxicity in order to define the specific neuronal targets related to its acute toxicity in insects and mammals. Although toxic to mosquitoes (LD₅₀ ca. 1.5 µg/mg), DEET was a poor acetylcholinesterase inhibitor (<10% inhibition), even at a concentration of 10 mM. IC₅₀ values for DEET against Drosophila melanogaster, Musca domestica, and human acetylcholinesterases were 6–12 mM. Neurophysiological recordings showed that DEET had excitatory effects on the housefly larval central nervous system (EC₅₀: 120 µM), but was over 300-fold less potent than propoxur, a standard anticholinesterase insecticide. Phentolamine, an octopamine receptor antagonist, completely blocked the central neuroexcitation by DEET and octopamine, but was essentially ineffective against hyperexcitation by propoxur and 4-aminopyridine, a potassium channel blocker. DEET was found to illuminate the firefly light organ, a tissue utilizing octopamine as the principal neurotransmitter. Additionally, DEET was shown to increase internal free calcium via the octopamine receptors of Sf21 cells, an effect blocked by phentolamine. DEET also blocked Na⁺ and K⁺ channels in patch clamped rat cortical neurons, with IC₅₀ values in the micromolar range. These findings suggest DEET is likely targeting octopaminergic synapses to induce neuroexcitation and toxicity in insects, while acetylcholinesterase in both insects and mammals has low (mM) sensitivity to DEET. The ion channel blocking action of DEET in neurons may contribute to the numbness experienced after inadvertent application to the lips or mouth of humans.     

Dopamine counteracts octopamine signalling in a neural circuit mediating food response in C. elegans

Animals assess food availability in their environment by sensory perception and respond to the absence of food by changing hormone and neurotransmitter signals. However, it is largely unknown how the absence of food is perceived at the level of functional neurocircuitry. In Caenorhabditis elegans, octopamine is released from the RIC neurons in the absence of food and activates the cyclic AMP response element binding protein in the cholinergic SIA neurons. In contrast, dopamine is released from dopaminergic neurons only in the presence of food. Here, we show that dopamine suppresses octopamine signalling through two D2‐like dopamine receptors and the G protein Gi/o. The D2‐like receptors work in both the octopaminergic neurons and the octopamine‐responding SIA neurons, suggesting that dopamine suppresses octopamine release as well as octopamine‐mediated downstream signalling. Our results show that C. elegans detects the absence of food by using a small neural circuit composed of three neuron types in which octopaminergic signalling is activated by the cessation of dopamine signalling.     

Central nervous sensitization and dishabituation of reflex action in an insect by the neuromodulator octopamine

Habituation of excitatory synaptic inputs onto identified motor neurons of the locust metathoracic ganglion, driven electrically and by natural stimuli, was examined using intracellular recording. Rapid progressive reduction in amplitude of EPSPs from a variety of inputs onto fast-type motor neurons occurred. The habituated EPSPs were quickly dishabituated by iontophoretic release of octopamine from a microelectrode into the neuropilar region of presumed synaptic action. The zone within which release was effective for a given neuron was narrowly-defined. With larger amounts of octopamine applied at a sensitive site the EPSP became larger than normal, and in many instances action potentials were initiated by the sensitized response. Very small EPSPs onto a motor neuron, which were associated with proprioceptive feedback, and which were originally too small to be detected above the noise, were potentiated to a level of several mV by the iontophoresed octopamine. A DUM neuron (presumed to be octopaminergic) was found, whose direct stimulation was followed by a strong dishabituating and sensitizing action leading to spikes, of inputs to an identified flexor tibiae motor neuron. The action and its time course were closely similar to those evoked by octopamine iontophoresed into the neuropil in the region of synaptic inputs to the motor neuron. It is concluded that DUM (octopaminergic) neurons exert large potentiating actions on central neuronal excitatory synaptic transmission in locusts.     

Odorant receptor co-receptor Orco is upregulated by methyl eugenol in male Bactrocera dorsalis (Diptera: Tephritidae)

Bactrocera dorsalis is a destructive fruit-eating pest that causes severe economic damage to the fruit and vegetable industry. Methyl eugenol (ME) has been widely used as an effective sexual attractant for male fruit flies through olfactory perception. However, the molecular mechanism underlying the olfactory perception of ME remains unknown. Here, we report the characterization and functional analysis of a newly discovered cDNA that encodes a Drosophila melanogaster odorant receptor co-receptor Orco ortholog in B. dorsalis. qRT-PCR analysis revealed that it was abundantly expressed in the antenna of adult B. dorsalis. Notably, Orco was upregulated by ME in the antenna of male flies. Mature males of B. dorsalis showed significant taxis toward ME within 0.5h, and Orco was significantly upregulated in the attracted adults within the same period. Silencing Orco through the ingestion of dsRNA reduced the attractive effects of ME. These data suggest that Orco may play an essential role in ME attraction in the olfactory signal transduction pathway.     

Molecular Clock Regulates Daily α1–2-Fucosylation of the Neural Cell Adhesion Molecule (NCAM) within Mouse Secondary Olfactory Neurons

The circadian clock regulates various behavioral and physiological rhythms in mammals. Circadian changes in olfactory functions such as neuronal firing in the olfactory bulb (OB) and olfactory sensitivity have recently been identified, although the underlying molecular mechanisms remain unknown. We analyzed the temporal profiles of glycan structures in the mouse OB using a high-density microarray that includes 96 lectins, because glycoconjugates play important roles in the nervous system such as neurite outgrowth and synaptogenesis. Sixteen lectin signals significantly fluctuated in the OB, and the intensity of all three that had high affinity for α1–2-fucose (α1–2Fuc) glycan in the microarray was higher during the nighttime. Histochemical analysis revealed that α1–2Fuc glycan is located in a diurnal manner in the lateral olfactory tract that comprises axon bundles of secondary olfactory neurons. The amount of α1–2Fuc glycan associated with the major target glycoprotein neural cell adhesion molecule (NCAM) varied in a diurnal fashion, although the mRNA and protein expression of Ncam1 did not. The mRNA and protein expression of Fut1, a α1–2-specific fucosyltransferase gene, was diurnal in the OB. Daily fluctuation of the α1–2Fuc glycan was obviously damped in homozygous Clock mutant mice with disrupted diurnal Fut1 expression, suggesting that the molecular clock governs rhythmic α1–2-fucosylation in secondary olfactory neurons. These findings suggest the possibility that the molecular clock is involved in the diurnal regulation of olfaction via α1–2-fucosylation in the olfactory system.     

Subunit contributions to insect olfactory receptor function: channel block and odorant recognition

Insect olfactory receptors are heteromeric ligand-gated ion channels composed of at least one common subunit (Orco) and at least one subunit that confers odorant specificity. Little is known about how individual subunits contribute to the structure and function of the olfactory receptor complex. We expressed insect olfactory receptors in Xenopus oocytes to investigate 2 functional features, ion channel block and odorant recognition. The sensitivity of Drosophila olfactory receptors to inhibition by ruthenium red, a cation channel blocker, varied widely when different specificity subunits were present, suggesting that the specificity subunits contribute to the structure of the ion pore. Olfactory receptors formed by Dmel\Or35a and Orco subunits from several different species displayed highly similar odorant response profiles, suggesting that the Orco subunit does not contribute to the structure of the odorant-binding site. We further explored odorant recognition by conducting a detailed examination of the odorant specificity Dmel\Or67a + Dmel\Orco, a receptor that responds to aromatic structures. This screen identified agonists, partial agonists, and an antagonist of Dmel\Or67a + Dmel\Orco. Our findings favor specific subunit arrangements within the olfactory receptor complex and provide a preliminary odorophore for an olfactory receptor, offering a useful foundation for future exploration of insect olfactory receptor structure.     

The Odorant Receptor Co-Receptor from the Bed Bug,Cimex lectularius L

Recently, the bed bug, Cimex lectularius L. has re-emerged as a serious and growing problem in many parts of the world. Presence of resistant bed bugs and the difficulty to eliminate them has renewed interest in alternative control tactics. Similar to other haematophagous arthropods, bed bugs rely on their olfactory system to detect semiochemicals in the environment. Previous studies have morphologically characterized olfactory organs of bed bugs’ antenna and have physiologically evaluated the responses of olfactory receptor neurons (ORNs) to host-derived chemicals. To date, odorant binding proteins (OBPs) and odorant receptors (ORs) associated with these olfaction processes have not been studied in bed bugs. Chemoreception in insects requires formation of heteromeric complexes of ORs and a universal OR coreceptor (Orco). Orco is the constant chain of every odorant receptor in insects and is critical for insect olfaction but does not directly bind to odorants. Orco agonists and antagonists have been suggested as high-value targets for the development of novel insect repellents. In this study, we have performed RNAseq of bed bug sensory organs and identified several odorant receptors as well as Orco. We characterized Orco expression and investigated the effect of chemicals targeting Orco on bed bug behavior and reproduction. We have identified partial cDNAs of six C. lectularius OBPs and 16 ORs. Full length bed bug Orco was cloned and sequenced. Orco is widely expressed in different parts of the bed bug including OR neurons and spermatozoa. Treatment of bed bugs with the agonist VUAA1 changed bed bug pheromone-induced aggregation behavior and inactivated spermatozoa. We have described and characterized for the first time OBPs, ORs and Orco in bed bugs. Given the importance of these molecules in chemoreception of this insect they are interesting targets for the development of novel insect behavior modifiers.     

Octopamine Neuromodulation Regulates Gr32a-Linked Aggression and Courtship Pathways in Drosophila Males

Chemosensory pheromonal information regulates aggression and reproduction in many species, but how pheromonal signals are transduced to reliably produce behavior is not well understood. Here we demonstrate that the pheromonal signals detected by Gr32a-expressing chemosensory neurons to enhance male aggression are filtered through octopamine (OA, invertebrate equivalent of norepinephrine) neurons. Using behavioral assays, we find males lacking both octopamine and Gr32a gustatory receptors exhibit parallel delays in the onset of aggression and reductions in aggression. Physiological and anatomical experiments identify Gr32a to octopamine neuron synaptic and functional connections in the suboesophageal ganglion. Refining the Gr32a-expressing population indicates that mouth Gr32a neurons promote male aggression and form synaptic contacts with OA neurons. By restricting the monoamine neuron target population, we show that three previously identified OA-Fru^M neurons involved in behavioral choice are among the Gr32a-OA connections. Our findings demonstrate that octopaminergic neuromodulatory neurons function as early as a second-order step in this chemosensory-driven male social behavior pathway.     

The PM1 neurons,movement sensitive centrifugal visual brain neurons in the locust: anatomy,physiology, and modulation by identified octopaminergic neurons

The locust’s optic lobe contains a system of wide-field, multimodal, centrifugal neurons. Two of these cells, the protocerebrum-medulla-neurons PM4a and b, are octopaminergic. This paper describes a second pair of large centrifugal neurons (the protocerebrum-medulla-neurons PM1a and PM1b) from the brain of Locusta migratoria based on intracellular cobalt fills, electrophysiology, and immunocytochemistry. They originate and arborise in the central brain and send processes into the medulla of the optic lobe. Double intracellular recording from the same cell suggests input in the central brain and output in the optic lobe. The neurons show immunoreactivity to gamma-amino-butyric acid and its synthesising enzyme, glutamate decarboxylase. The PM1 cells are movement sensitive and show habituation to repeated visual stimulation. Bath application of octopamine causes the response to dishabituate. A very similar effect is produced by electrical stimulation of one of an octopaminergic PM4 neuron. This effect can be blocked by application of the octopamine antagonists, mianserin and phentolamine. This readily accessible system of four wide-field neurons provides a system suitable for the investigation of octopaminergic effects on the visual system at the cellular level.     

Fused in Sarcoma (FUS) Protein Lacking Nuclear Localization Signal (NLS) and Major RNA Binding Motifs Triggers Proteinopathy and Severe Motor Phenotype in Transgenic Mice

Dysfunction of two structurally and functionally related proteins, FUS and TAR DNA-binding protein of 43 kDa (TDP-43), implicated in crucial steps of cellular RNA metabolism can cause amyotrophic lateral sclerosis (ALS) and certain other neurodegenerative diseases. The proteins are intrinsically aggregate-prone and form non-amyloid inclusions in the affected nervous tissues, but the role of these proteinaceous aggregates in disease onset and progression is still uncertain. To address this question, we designed a variant of FUS, FUS 1–359, which is predominantly cytoplasmic, highly aggregate-prone, and lacks a region responsible for RNA recognition and binding. Expression of FUS 1–359 in neurons of transgenic mice, at a level lower than that of endogenous FUS, triggers FUSopathy associated with severe damage of motor neurons and their axons, neuroinflammatory reaction, and eventual loss of selective motor neuron populations. These pathological changes cause abrupt development of a severe motor phenotype at the age of 2.5–4.5 months and death of affected animals within several days of onset. The pattern of pathology in transgenic FUS 1–359 mice recapitulates several key features of human ALS with the dynamics of the disease progression compressed in line with shorter mouse lifespan. Our data indicate that neuronal FUS aggregation is sufficient to cause ALS-like phenotype in transgenic mice.     

Increase in membrane surface expression and phosphorylation of TRPC3 related to olfactory dysfunction in α‐synuclein transgenic mice

Olfactory impairment is an initial non‐motor symptom of Parkinson''s disease that causes the deposition of aggregated α‐synuclein (α‐syn) in olfactory neurons. Transient receptor potential canonical (TRPC) channels are a diverse group of non‐selective Ca²⁺ entry channels involved in the progression or pathogenesis of PD via Ca²⁺ homeostatic regulation. However, the relationship between TRPC and α‐syn pathology in an olfactory system remains unclear. To address this issue, we assessed the olfactory function in α‐syn transgenic mice. In contrast with control mice, the transgenic mice exhibited impaired olfaction, TRPC3 activation and apoptotic neuronal cell death in the olfactory system. Similar results were observed in primary cultures of olfactory neurons, that is TRPC3 activation, increasing intracellular Ca²⁺ concentration and apoptotic cell death in the α‐syn‐overexpressed neurons. These changes were significantly attenuated by TRPC3 knockdown. Therefore, our findings suggest that TRPC3 activation and calcium dyshomeostasis play a key role in α‐syn‐induced olfactory dysfunction in mice.     

The olfactory co-receptor Orco from the migratory locust (Locusta migratoria) and the desert locust (Schistocerca gregaria): identification and expression pattern

In locusts, olfaction plays a crucial role for initiating and controlling behaviours, including food seeking and aggregation with conspecifics, which underlie the agricultural pest capacity of the animals. In this context, the molecular basis of olfaction in these insects is of particular interest. Here, we have identified genes of two orthopteran species, Locusta migratoria and Schistocera gregaria, which encode the olfactory receptor co-receptor (Orco). It was found that the sequences of LmigOrco and SgreOrco share a high degree of identity to each other and also to Orco proteins from different insect orders. The Orco-expressing cells in the antenna of S. gregaria and L. migratoria were visualized by in situ hybridization. Orco expression could be assigned to clusters of cells in sensilla basiconica and few cells in sensilla trichodea, most likely representing olfactory sensory neurons. No Orco-positive cells were detected in sensilla coeloconica and sensilla chaetica. Orco expression was found already in all nymphal stages and was verified in some other tissues which are equipped with chemosensory hairs (mouthparts, tarsi, wings). Together, the results support the notion for a decisive role of Orco in locust olfaction.     

Neural sex modifies the function of a C. elegans sensory circuit

Though sex differences in animal behavior are ubiquitous, their neural and genetic underpinnings remain poorly understood. In particular, the role of functional differences in the neural circuitry that is shared by both sexes has not been extensively investigated. We have addressed these issues with C. elegans olfaction, a simple innate behavior mediated by sexually isomorphic neurons. Though males respond to the same olfactory attractants as do hermaphrodites, we find that each sex has a characteristic repertoire of olfactory preferences. These are not secondary to other sex-specific behaviors and do not require signaling from the gonad. Sex-specific olfactory preferences are controlled by tra-1, the master regulator of C. elegans sexual differentiation. Moreover, the genetic masculinization of neurons in an otherwise wild-type hermaphrodite is sufficient to switch the sexual phenotype of olfactory preference behavior. These studies reveal novel and unexpected sex differences in a C. elegans sensory behavior that is exhibited by both sexes. Our results indicate that these differences are a function of the chromosomally determined sexual identity of shared neural circuitry.     

Kinesin-2 transports Orco into the olfactory cilium of Drosophila melanogaster at specific developmental stages

The cilium, the sensing centre for the cell, displays an extensive repertoire of receptors for various cell signalling processes. The dynamic nature of ciliary signalling indicates that the ciliary entry of receptors and associated proteins must be regulated and conditional. To understand this process, we studied the ciliary localisation of the odour-receptor coreceptor (Orco), a seven-pass transmembrane protein essential for insect olfaction. Little is known about when and how Orco gets into the cilia. Here, using Drosophila melanogaster, we show that the bulk of Orco selectively enters the cilia on adult olfactory sensory neurons in two discrete, one-hour intervals after eclosion. A conditional loss of heterotrimeric kinesin-2 during this period reduces the electrophysiological response to odours and affects olfactory behaviour. We further show that Orco binds to the C-terminal tail fragments of the heterotrimeric kinesin-2 motor, which is required to transfer Orco from the ciliary base to the outer segment and maintain within an approximately four-micron stretch at the distal portion of the ciliary outer-segment. The Orco transport was not affected by the loss of critical intraflagellar transport components, IFT172/Oseg2 and IFT88/NompB, respectively, during the adult stage. These results highlight a novel developmental regulation of seven-pass transmembrane receptor transport into the cilia and indicate that ciliary signalling is both developmentally and temporally regulated.

Jana, Dutta, Jain et al., show that the odour-receptor coreceptor only enters the cilia expressed on olfactory sensory neurons at specified developmental stages requiring heterotrimeric kinesin-2. The motor binds to the coreceptor and plays a crucial role in localising them to a compact, environment-exposed domain at the ciliary outer-segment.     

EGFR signaling modulates synaptic connectivity via Gurken

Synaptic target selection is critical for establishing functional neuronal circuits. The mechanisms regulating target selection remain incompletely understood. We describe a role for the EGF receptor and its ligand Gurken in target selection of octopaminergic Type II neurons in the Drosophila neuromuscular system. Mutants in happyhour, a regulator of EGFR signaling, form ectopic Type II neuromuscular junctions. These ectopic innervations are due to inappropriate target selection. We demonstrate that EGFR signaling is necessary and sufficient to inhibit synaptic target selection by these octopaminergic Type II neurons, and that the EGFR ligand Gurken is the postsynaptic, muscle-derived repulsive cue. These results identify a new pathway mediating cell-type and branch-specific synaptic repulsion, a novel role for EGFR signaling in synaptic target selection, and an unexpected role for Gurken as a muscle-secreted repulsive ligand.