InteractiveFly: GeneBrief

Octopamine β3 receptor: Biological Overview | References

In Drosophila, pulsed production of the steroid hormone ecdysone plays a pivotal role in developmental transitions such as metamorphosis. Ecdysone production is regulated in the prothoracic gland (PG) by prothoracicotropic hormone (PTTH) and insulin-like peptides (Ilps). This study shows that monoaminergic autocrine regulation of ecdysone biosynthesis in the PG is essential for metamorphosis. PG-specific knockdown of a monoamine G protein-coupled receptor, β3-octopamine receptor (Octβ3R), resulted in arrested metamorphosis due to lack of ecdysone. Knockdown of tyramine biosynthesis genes expressed in the PG caused similar defects in ecdysone production and metamorphosis. Moreover, PTTH and Ilps signaling were impaired by Octβ3R knockdown in the PG, and activation of these signaling pathways rescued the defect in metamorphosis. Thus, monoaminergic autocrine signaling in the PG regulated ecdysone biogenesis in a coordinated fashion on activation by PTTH and Ilps. The study proposes that monoaminergic autocrine signaling acts downstream of a body size checkpoint that allows metamorphosis to occur when nutrients are sufficiently abundant (Ohhara, 2015).

In many animal species, the developmental transition is a well-known biological process in which the organism alters its body morphology and physiology to proceed from the juvenile growth stage to the adult reproductive stage. For example, in mammals, puberty causes a drastic change from adolescent to adulthood, whereas in insects, metamorphosis initiates alteration of body structures to produce sexually mature adults, a process accompanied by changes in habitat and behavior. These developmental transitions are primarily regulated by steroid hormones, production of which is regulated coordinately by developmental timing and nutritional conditions. How these processes are precisely regulated in response to developmental and environ mental cues is a longstanding question in biology (Ohhara, 2015).

In holometabolous insects, the steroid hormone ecdysone plays a pivotal role in metamorphosis. In Drosophila, metamorphic development from the third-instar larva into the adult, through the prepupa and pupa, initiates 90-96 h after hatching (hAH) at 25°C under a standard culture condition. At the onset of the larval-prepupal transition, ecdysone is produced in the prothoracic gland (PG) and then converted into its active form, 20-hydroxyecdysone (20E), in the peripheral organs. The activities of 20E terminate larval development and growth and initiates metamorphosis. Ecdysone biosynthesis is regulated in the PG by neuropeptides, enabling modulation of the timing of 20E pulses during development. The best-known stimulator of ecdysone biosynthesis is prothoracico-tropic hormone (PTTH), which is produced by neurons in the CNS. PTTH activates the receptor tyrosine kinase Torso in the PG to stimulate expression of ecdysone biosynthetic genes through the Ras85D/Raf/MAPK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. Insulin-like peptides (Ilps), members of another class of neuron-derived factors, activate PI3K in the PG, resulting in production of ecdysone biosynthetic proteins. The Activin/transforming growth factor-β (TGF-β) signaling pathway is also required in the PG for the expression of PTTH and Ilps receptors, although to date it remains unclear which organ produces the ligand that acts on the PG (Ohhara, 2015).

In addition to these neuropeptides, the larval-prepupal transition is modulated by environmental cues such as nutritional conditions that influence larval body size. For example, at 56 hAH, early third-instar larvae attain the minimal viable weight (MVW), at which sufficient nutrition is stored in larvae to ensure their survival through metamorphosis. After attaining MVW, larvae pass another checkpoint, critical weight (CW), defined as the minimum larval size at which starvation no longer delays the larval-prepupal transition. In Drosophila, both checkpoints occur almost simultaneously, making it difficult to distinguish them. However, CW is regarded as a body size checkpoint that initiates metamorphosis and is therefore believed to ultimately modulate ecdysone production in the PG. However, its downstream effectors and signaling pathway remain elusive (Ohhara, 2015).

Based on data obtained in Manduca and Bombyx, a G protein-coupled receptor (GPCR) has long been postulated to be essential for ecdysone biosynthesis in the PG. However, this GPCR and its ligand have not yet been identified. This study shows that monoaminergic autocrine signaling through a GPCR, β3-octopamine receptor (Octβ3R), plays an essential role in ecdysone biosynthesis to execute the larval-prepupal transition. Octβ3R is also required for activation of PTTH and Ilps signaling. It is proposed that this autocrine system acts downstream of the CW checkpoint to allow the larval-prepupal transition. It is speculated that monoamines play an evolutionarily conserved role in the regulation of steroid hormone production during developmental transitions (Ohhara, 2015).

Previously studies have shown that the GPCR Octβ3R is expressed in multiple larval tissues, including the PG (Ohhara, 2012). To determine whether Octβ3R is involved in ecdysone biosynthesis and metamorphosis, RNAi was used to knock down Octβ3R function specifically in the PG, using the Gal4-upstream activation sequence (UAS) system. Two different UAS-Octβ3RRNAi constructs targeting distinct regions of the Octβ3R mRNA (Octβ3R^RNAi-1 and Octβ3R^RNAi-2) were used to knock down Octβ3R in the PG with the help of a phantom (phm)-22-Gal4 driver. Strikingly, larvae expressing Octβ3RRNAi in the PG never developed into adult flies, and 96% of phm>Octβ3R^RNAi-1 animals and 34% of phm>Octβ3R^RNAi-2 animals arrested at the larval stage. When UAS-dicer2 was introduced into phm>Octβ3R^RNAi-2 larvae (phm>Octβ3R^RNAi-2+dicer2) to increase RNAi activity, all of these animals arrested at the larval stage. Using in situ hybridization, a significant reduction was confirmed in the Octβ3R mRNA levels in the PG of knockdown animals relative to control larvae. These data suggest that Octβ3R expression in the PG is essential for executing the larval-prepupal transition in metamorphosis (Ohhara, 2015).

Because a similar defect in the larval-prepupal transition occurs in ecdysone-deficient larvae, it was hypothesized that the Octβ3R knockdown phenotype was due to lack of ecdysone production. Consistent with this idea, the 20E titer was much lower in phm>Octβ3R^RNAi-1 larvae than in control larvae just before the larval-prepupal transition (90 hAH). Moreover, administration of 20E by feeding rescued the defect in the larval- prepupal transition caused by Octβ3R knockdown. When phm>Octβ3R^RNAi-1 and phm>Octβ3R^RNAi-2+dicer2 larvae were cultured on media containing 20E (1 mg/mL) from 48 hAH onward, approximately half of them developed to the prepupal stage, compared with only 2-3% of larvae not fed 20E. Thus, PG-specific loss of Octβ3R activity causes an arrest in the larval-prepupal transition due to lack of ecdysone (Ohhara, 2015).

Ecdysone is synthesized in the PG from dietary cholesterol through the action of seven ecdysone biosynthetic genes (neverland, spookier, shroud, Cyp6t3, phantom, disembodied, and shadow). Quantitative RT- PCR (qPCR) was performed to investigate whether loss of Octβ3R function affects expression of these genes in the PG. In control larvae, expression of these genes increased dramatically between 72 and 96 hAH, when the larval-prepupal transition occurs. By contrast, in phm>Octβ3R^RNAi-1 and phm>Octβ3R^RNAi-2+dicer2 larvae, the expression of all of these genes was significantly reduced relative to control larvae at 96 hAH. The reduced expression of ecdysone biosynthetic genes in the PG was confirmed by in situ hybridization. Furthermore, immunostaining revealed that Neverland, Shroud, Phantom, Disembodied, and Shadow protein levels were reduced in the PG of phm>Octβ3R^RNAi-1 and phm>Octβ3R^RNAi-2+dicer2 larvae. Taken together, these data show that Octβ3R function is required in the PG for proper expression of ecdysone biosynthetic genes (Ohhara, 2015).

Octβ3R is thought to be activated by octopamine and tyramine binding. Octopamine is synthesized from tyramine by tyramine β-hydroxylase (Tbh), and tyramine is synthesized from tyrosine by tyrosine decarboxylase (Tdc). In Drosophila, two Tdc genes (Tdc1 and Tdc2) and one Tbh gene have been identified, and all of them are expressed in the larval CNS. Tdc1, Tdc2, and Tbh are also expressed in the PG. Furthermore, octopamine and tyramine were detected in the PG by immunostaining. Thus, octopamine and/or tyramine synthesized in the PG may activate Octβ3R in an autocrine manner to induce ecdysone production (Ohhara, 2015).

To test this, PG-specific knockdowns of Tdc1, Tdc2, and Tbh were generated. To knock down Tdc2, two constructs targeting distinct regions of the Tdc2 transcript (Tdc2^RNAi-1 and Tdc2^RNAi-2) were expressed along with dicer2 in the PG under the control of the phm-22-Gal4 driver (phm > Tdc2^RNAi-1+dicer2 and phm > Tdc2^RNAi-2+dicer2). All phm > Tdc2^RNAi-1+dicer2 larvae arrested at the larval stage, and phm > Tdc2^RNAi-2+dicer2 larvae were significantly delayed at the larval-prepupal transition, relative to control animals. Tdc2 mRNA level was reduced in the ring gland (RG) containing the PG in both sets of knockdown animals, as demonstrated by qPCR. Moreover, octopamine and tyramine production in the PG was impaired by Tdc2 knockdown. By contrast, Tdc1 knockdown (phm > Tdc1RNAi+dicer2) caused only a subtle delay in the larval-prepupal transition and had no detectable effect on octopamine or tyramine production. These results suggest that Tdc2 is the predominant Tdc regulating octopamine and tyramine biosynthesis in the PG and the larval-prepupal transition. Contrary to these findings, a null mutation in Tdc2 does not affect metamorphosis, and these mutant flies are viable. Thus, PG-specific knockdown causes a stronger phenotype than complete loss of Tdc2 activity in whole animals. A similar situation has been reported in regulation of metamorphosis by Activin signaling. These phenomena can be explained by a model in which some compensatory changes in other mutant tissues rescue the PG-specific knockdown phenotype in null-mutant animals (Ohhara, 2015).

PG-specific Tdc2 knockdown caused a reduction in larval 20E concentration. Therefore, whether feeding 20E to Tdc2 knockdown larvae would rescue the larval- prepupal transition defect was examined. To this end, phm > Tdc2^RNAi-1+ dicer2 and phm > Tdc2^RNAi-2+dicer2 larvae were cultured in media with or without 20E (1 mg/mL) from 48 hAH onward. Approximately 40% of the 20E-fed phm > Tdc2^RNAi-1+dicer2 larvae developed to the prepupal stage, whereas none of those larvae grown on control media progressed beyond the larval stage. Furthermore, the delay in the larval-prepupal transition in phm > Tdc2^RNAi-2+dicer2 larvae was rescued by 20E feeding. These results indicate that the defect in the larval-prepupal transition in Tdc2 knockdown animals results from a lack of 20E production. Thus, octopamine/ tyramine synthesized in the PG appears to activate Octβ3R in an autocrine manner to execute the larval-prepupal transition by regulating ecdysone production (Ohhara, 2015).

To determine which Octβ3R ligand is responsible for this autocrine signaling, Tbh was knocked down in the PG to prevent conversion of tyramine into octopamine. To knock down Tbh, two constructs targeting distinct regions of the Tbh transcript (Tbh^RNAi-1 and Tbh^RNAi-2) were expressed along with dicer2 under the control of phm-22-Gal4 (phm > Tbh^RNAi-1+ dicer2 and phm > Tbh^RNAi-2+dicer2). Although the Tbh knockdown caused a reduction in octopamine production in the PG, these larvae did not exhibit any obvious defects in the larval-prepupal transition or subsequent metamorphosi. These data suggest that tyramine, rather than octopamine, is the Octβ3R ligand that activates ecdysone production in the PG (Ohhara, 2015).

Because ecdysone biosynthesis in the PG is under the control of Ilps and PTTH signaling, it was next examined whether Octβ3R function is required to activate these signaling pathways. To detect Ilps signaling activity, a pleckstrin-homology domain fused to GFP (PH-GFP), which is recruited to the plasma membrane when insulin signaling is activated, was used. In the PG cells of control larvae, PH-GFP was only weakly localized to the plasma membrane at 48 hAH, whereas its membrane localization became increasingly evident at 60, 84, and 90 hAH. By contrast, in PG cells of phm>Octβ3R^RNAi-1 larvae, the tight localization of PH-GFP to the plasma membrane was no longer detectable, indicating that activation of Ilps signaling had been disrupted. Moreover, overexpression of a constitutively active form of the Ilps receptor InR (InRCA) was able to rescue the larval arrest in phm>Octβ3R^RNAi-1 animals. Next, immunostaining was performed of the diphosphorylated form of ERK (dpERK), a downstream signaling component of the PTTH pathway. dpERK expression was found to be very weak at 48 hAH, but was activated in the PG of control larvae at 60, 84, and 90 hAH; by contrast, this activation was reduced in the PG of phm>Octβ3R^RNAi-1 larvae. Expression of a constitutively active form of another downstream PTTH signaling component, Ras (RasV12), rescued the larval-prepupal transition defect in phm>Octβ3R^RNAi-1 animals. These results show that Octβ3R function is required to activate Ilps and PTTH signaling in the PG and that these signaling pathways execute the larval-prepupal transition. Although activation of both the Ilps and PTTH signaling pathways requires Activin/TGFβ signaling in the PG, expression of a constitutively active form of the Activin/ TGFβ receptor Baboon (BaboCA) failed to rescue the larval-prepupal transition defect in phm>Octβ3R^RNAi-1 animals. This observation suggests that Octβ3R acts downstream or independent of Activin/TGFβ signaling to regulate Ilps and PTTH signaling in the PG (Ohhara, 2015).

The observations described above demonstrate that phm>Octβ3RRNAi affects Ilps and PTTH signaling in the PG as early as 60 hAH, raising the question of when Octβ3R function is required in the PG for execution of the larval-prepupal transition. To address this issue, the Gal80ts and Gal4/UAS system, which restricts expression of Octβ3R dsRNA in the PG at 18^oC, but allows its expression at 28^oC, was used. The results of temperature upshift and downshift experiments revealed that the larval-prepupal transition was impaired only when Octβ3R dsRNA was expressed in the PG at around 60 hAH. Notably, 60 hAH is the critical period during which larvae attain CW under nutrient-rich conditions. As noted above, when larvae are starved before attainment of CW, they are unable to transit into the prepupal stage. By contrast, starved larvae can successfully transit to prepupal/pupal stage without developmental delay once they have attained CW by growing beyond the critical period (~56 hAH) under nutrient-rich conditions in standard Drosophila medium. Thus, it is hypothesized that Octβ3R signaling acts downstream of the body-size checkpoint, or attainment of CW, to allow the larval-prepupal transition (Ohhara, 2015).

Several lines of evidence support this hypothesis. First, Octβ3R function is required for activation of Ilps and PTTH signaling detected in the PG at 60 hAH. By contrast, at 48 hAH, before the attainment of CW, neither signaling pathway is active in the PG. Second, Ilps and PTTH signaling was not activated in the PG when the larvae were starved from 48 hAH onward (early starvation), whereas these signaling pathways were active when the larvae were starved after 60 hAH (late starvation). Finally, a ligand for Octβ3R, tyramine, was detectable in the PG at 60 hAH, but decreases after this stage under a nutrient-rich condition. This decrease in tyramine was abrogated by early starvation but not by late starvation. Assuming that this decrease in tyramine in the PG is due to its secretion from PG cells, it is reasonable to propose that attainment of CW causes tyramine secretion from the PG at around 60 hAH, which in turn activates Octβ3R to regulate the Ilps and PTTH pathways, leading to the larval-prepupal transition (Ohhara, 2015).

This study demonstrates that monoaminergic regulation plays a pivotal role in ecdysone biosynthesis to induce metamorphosis and that Octβ3R acts as an upstream regulator essential for the Ilps and PTTH signaling. In addition, the data indicate that Octβ3R ligands are produced in the PG to stimulate ecdysone biosynthesis in an autocrine manner. Autocrine signaling has been proposed to mediate the community effect, in which identical neighboring cells are coordinated in their stimulation and maintenance of cell type-specific gene expression and their differentiation, as observed in muscle development of amphibian embryos. Thus, it is proposed that monoaminergic autocrine signaling among PG cells acts to increase their responsiveness to Ilps and PTTH, thereby allowing coordinated ex- pression of ecdysone biosynthetic genes within a time window following exposure to neuropeptides (Ohhara, 2015).

These findings raise the larger question of whether monoamine acts as part of an evolutionarily conserved mechanism of steroid hormone production. In vertebrates, there is limited evidence of monoaminergic regulation of steroid hormone biosynthesis. For example, in cultured adrenal glands, catecholamine stimulates the biosynthesis of the steroid hormone cortisol in a paracrine manner to elicit a stress reaction. Another example is the Leydig cells of the mammalian testes, in which the steroid hormone testosterone is produced mainly in response to pituitary gonadotropin. However, catecholamine signaling through β-adrenergic receptors, the orthologs of Octβ3R, also promotes the production of testosterone from cultured fetal Leydig cells, which may be the site of catecholamine synthesis in the fetal and mature human testes. Thus, monoamines may play a conserved role in modulating and/or stimulating steroid hormone production during physiological and developmental transitions (Ohhara, 2015).

Genetic and neurobiological analyses of the noradrenergic-like system in vulnerability to sugar overconsumption using a Drosophila model

Regular overconsumption of sugar is associated with obesity and type-2 diabetes, but how genetic factors contribute to variable sugar preferences and intake levels remains mostly unclear. This study provides evidence for the usefulness of a Drosophila larva model to investigate genetic influence on vulnerability to sugar overconsumption. Using genetic and RNA interference approaches, this study shows that the activity of the Oamb gene, which encodes a receptor for octopamine (OA, the invertebrate homologue of norepinephrine), plays a major role in controlled sugar consumption. Furthermore, Oamb appears to suppress sugar food intake in fed larvae in an acute manner, and neurons expressing this Oamb receptor do not overlap with neurons expressing Octbeta3R, another OA receptor previously implicated in hunger-driven exuberant sugar intake. Together, these results suggest that two separate sub-circuits, defined by Oamb and Octbeta3R respectively, co-regulate sugar consumption according to changes in energy needs. It is proposed that the noradrenergic-like system defines an ancient regulatory mechanism for prevention of sugar overload (Branch, 2017).

This study has shown that two of the four OA receptors encoded by the Drosophila genome mediate the dual role of the OA system in modulation of feeding of readily available sugar food under different motivational states. An α-adrenergic-like receptor Oamb is acutely required for prevention of sugar overconsumption in fed larvae, while a β-adrenergic-like receptor Octβ3R is required for hunger-driven responses to the sugar food. These findings suggest that the adrenergic-like system of invertebrate animals is a crucial regulator that links the motivational state to the adaptive consumption of sugar, a vital energy source (Branch, 2017).

Sugar food preference is known to vary among individuals, and understanding of how genetic factors contribute to such variations remain limited. This study has shown that functional deficiency of the Oamb gene caused significant increases in the sugar food consumption in fed larvae. These results raise the possibility that mutations in an array of genes involved in the OA/Oamb pathway may also have similar effects on sugar food consumption. Therefore, these findings suggest that the fly larva may be a useful platform for investigating the contributions of genetic factors to variations in sugar consumption among individual animals. It would also be interesting to test whether genetic variations that affect the function of norepinephrine system may underlie the genetic predisposition to crave for sugar-rich food in mammals (Branch, 2017).

A previous study provided evidence for a potential interaction between the OA/Oamb- and OA/Octβ3R-mediated sub-circuits in modulation of sugar consumption by fly larvae (Zhang, 2013). It has shown that two separate subsets of OA neurons (named VUM1 and VUM2, respectively) in the hindbrain-like region are required for the control of sugar food ingestion. Targeted lesioning of VUM1 resulted in sugar overconsumption in fed larvae, while targeted lesioning of VUM2 attenuated Octβ3R-dependent feeding of sugar food in hungry larvae. Further, targeted lesioning of VUM2 also attenuated Octβ3R-dependent feeding response to sugar food. However, how VUM1 and VUM2 neurons functionally interact with each other remains unclear. This work supports the notion that VUM1 neurons are acutely active in fed larvae but silenced under prolonged food deprivation. In fed larvae, VUM1 may indirectly suppress a VUM2-dependent sub-circuit through its signaling to Oamb neurons. It is possible that the VUM1/Oamb neuronal pathway may exert the inhibitory effect on the VUM2/Octβ3R neuronal pathway at the level of the Octβ3R neurons or their downstream targets. Further experiments will be needed to determine how the OA/Oamb and OA/Octβ3R sub-circuits interact to co-regulate sugar consumption under different motivational states (Branch, 2017).

Carbohydrates are vital energy sources to animals across evolution. Despite considerable evolutionary divergence, the control mechanisms for carbohydrate intake in insects and mammals may share similar molecular and neural mechanisms. For example, OA neurons from the hindbrain-like SOG region are known to be associated with sugar sensation in insects. Treatment of OA promotes honey bee's feeding response toward sucrose, and is able to increase the reward value of food resources. It has also been reported that OA is necessary and can even replace sugar stimuli in forming appetitive olfactory memories in Drosophila1. Similarly, a group of norepinephrine (the vertebrate counterpart of OA) neurons in the brainstem of rats are responsive to glucose level required for regulating carbohydrates-specific food ingestion (Branch, 2017).

It is proposed that precise control of feeding is achieved through different affinities between agonists and different receptors, and the relative activity level of α1 and α2 receptor neurons determines the feeding consequences. In rats, antagonistic effects of altering food intake are mediated through different downstream receptor neurons located in the paraventricular nucleus of hypothalamus. NE signaling promotes feeding through α1 receptors, while its activation of α2 receptors inhibits food intake. In Drosophila larvae, this study has also identified two separate OA circuits exerting opposite effects in regulating feeding. Similar to mammalian models, two different downstream receptors are found exhibiting antagonistic effects. Both 1.6-Oamb-GAL4 and 1.8-Octβ3R-GAL4 neurons are present in a larval brain region anterior to the OA neurons. It would be interesting to determine whether this region represents a functional equivalence of the mammalian hypothalamus. Furthermore, satiation status in rats affects an animal's feeding decisions by altering both NE release adrenoceptor levels. It is postulated that the OA system is also subject to modulation by endocrine hormones and nutrients levels, and it may define a key control site in the central nervous system where multi-sensory integration and feeding regulation takes place (Branch, 2017).

Octopamine-mediated circuit mechanism underlying controlled appetite for palatable food in Drosophila

The easy accessibility of energy-rich palatable food makes it difficult to resist food temptation. Drosophila larvae are surrounded by sugar-rich food most of their lives, raising the question of how these animals modulate food-seeking behaviors in tune with physiological needs. This study describes a circuit mechanism defined by neurons expressing tdc2-Gal4 (a tyrosine decarboxylase 2 promoter-directed driver) that selectively drives a distinct foraging strategy in food-deprived larvae. Stimulation of this otherwise functionally latent circuit in tdc2-Gal4 neurons was sufficient to induce exuberant feeding of liquid food in fed animals, whereas targeted lesions in a small subset of tdc2-Gal4 neurons in the subesophageal ganglion blocked hunger-driven increases in the feeding response. Furthermore, regulation of feeding rate enhancement by tdc2-Gal4 neurons requires a novel signaling mechanism involving the VEGF2-like receptor, octopamine, and its receptor. These findings provide fresh insight for the neurobiology and evolution of appetitive motivation (Zhang, 2013).

Four different OA receptors have been identified in Drosophila. To determine the downstream effectors of the OA feeding pathway, a mifepristone (RU486)-inducible pan-neural GS-elav-Gal4 driver was used to perform dsRNA-mediated conditional knockdown of individual OA receptor activity. Only disruption of the β-adrenergic–like Octβ3R receptor blocked starvation-induced larval mouth hook contraction rate increases. Unlike in tdc2Gal4/UAS-Kir2.1 larvae, oral introduction of OA failed to rescue the defect of feeding response in fasted GS-elav-Gal4/UAS-Octβ3RdsRNA larvae. Furthermore, Octβ3RMB04794 larvae containing a transposable element that disrupts Octβ3R are also deficient in the hunger-driven feeding response. These results suggest that OA and Octβ3R define a circuit mechanism that enhances feeding of liquid sugar media in fasted larvae (Zhang, 2013).

Modulation of feeding responses to food sources is heavily influenced by nutritional quality, taste, and the energy costs of foraging. The current findings suggest that Drosophila larvae have evolved a complex neural network to regulate appetitive motivations. In hungry fly larvae, OA neurons seem to mediate a specialized circuit that selectively promotes persistent feeding of readily ingestible sugar food. This OA circuit functions in parallel to the previously characterized mechanism coregulated by the fly insulin and NPY-like systems that drives feeding response to non-preferred solid food. Because food deprivation triggers simultaneous activation of both circuits, hungry larvae become capable of adaptively responding to diverse energy sources of high or low quality. It remains to be determined how OA signaling promotes persistent feeding response to liquid sugar food in hungry larvae. One possible scenario is that OA neurons in the SOG may be conditionally activated by gustatory cues associated with rich palatable food to promote appetitive motivation (Zhang, 2013).

This study has has provided evidence, at both molecular and neuronal levels, that the OA-mediated feeding circuit has two opposing effects on food motivation. When surrounded by liquid sugar media, the OA circuit is essential to prevent fed animals from excessive feeding. Because targeted lesions in VUM1 neurons caused excessive feeding response, these neurons may define an inhibitory subprogram within the OA feeding circuit. However, targeted lesions in VUM2 neurons attenuated hunger-induced increases of feeding response, suggesting that VUM2 neurons, along with the OA receptor Octβ3R, may define a subprogram that enhances feeding in fasted larvae. Several lines of evidence suggest that the VUM2 neuron-mediated subprogram may be suppressed by the VUM1 neuron-mediated subprogram. First, fed larvae with double lesions in both VUM1 and VUM2 neurons failed to display excessive feeding, suggesting that increased feeding response of fed larvae deficient for VUM1 neuronal signaling requires VUM2 neurons. Second, targeted lesions in VUM2 neurons of fed tdc2-Gal4/UAS- dTrpA1 larvae completely blocked the increased feeding response induced by genetic activation of tdc2-Gal4 neurons. Finally, the anatomical data also show that VUM1 and VUM2 neurons project to many common regions of the larval brain implicated in the control of feeding. Future work will be needed to determine whether VUM1 neurons inhibit directly or indirectly the activity of VUM2 neurons (Zhang, 2013).

Genetic and pharmacological evidence has been obtained for the critical role of OA in the regulation of acquiring readily accessible sugar media. OA has been reported to mediate diverse neurobiological functions including appetitive memory formation and modulation of the dance of honey bee foragers to communicate floral or sucrose rewards. It is postulated that the different OA receptors may mediate diverse OA-dependent behavioral responses to high-quality foods (Zhang, 2013).

Norepinephrine (NE), the vertebrate counterpart of OA, has been shown to promote ingestion of carbohydrate-rich food at the beginning of a natural feeding cycle. This feeding activity of NE resides in the paraventricular nucleus (PVN) of the feeding control center. In the PVN, α1 and α2 adrenergic receptors are organized in an antagonistic pattern. Activation of α1 receptor inhibits food intake, whereas activation of the α2 receptor stimulates food intake. The current results suggest that the insect OA system, like the NE system in mammals, exerts both positive and negative effects on the intake of preferred food. The activity of NE in PVN has been shown to antagonize that of 5-HT, which suppresses intake of carbohydrate- rich food. In Drosophila, 5-HT is also known to suppress feeding response. These findings suggest that the homeostatic control of intake of preferred food is likely mediated by a conserved neural network in flies and mammals (Zhang, 2013).

This study has identified a unique role of Pvr in physiological regulation of hunger-motivated feeding of preferred food (liquid sugar media). The feeding-related activity of the Pvr pathway involves two regulatory proteins, Drk and Ras, and oral introduction of OA restores the hunger-driven feeding response in tdc2-Gal4/ UAS-drkdsRNA larvae. Together, these results suggest that the Pvr pathway positively regulates OA release by tdc2-Gal4 neurons. Among the three identified ligands of Pvr, Pvf2 is enriched in the larval CNS. The current finding suggests that Pvf2 regulates the feeding-related activity of the Pvr pathway. It is possible that Pvf2 may transduce a metabolic stimulus to Pvr/tdc2-Gal4 neurons that signals the energy state of larvae. In the honey bee brain, OA neurons from the SOG have been reported to respond to sugar stimulation. Therefore, it would be interesting to test whether the Pvf2/ Pvr pathway is responsive to sugar stimuli (Zhang, 2013).

Previous studies have shown that the fly insulin and NPY-like systems coregulate hunger-elicited motivation to acquire solid sugar media. This study has now provided evidence that the fly VEGFR2- and NE-like systems control larval motivation to acquire liquid sugar media. These findings strongly suggest that the neural activities of different RTK systems play critical roles in different aspects of adaptive feeding decisions under various food and metabolic conditions. Therefore, further investigation of the mechanistic details of the food-related functions of RTK systems in the Drosophila model may provide novel insights into the neurobiology and evolution of appetitive control as well as pathophysiology of eating-related disorders (Zhang, 2013).

Expression of beta-adrenergic-like octopamine receptors during Drosophila development

An invertebrate biogenic amine, octopamine, plays diverse roles in multiple physiological processes (e.g. neurotransmitter, neuromodulator, and circulating neurohormone). Octopamine is thought to function by binding to G-protein-coupled receptors. In Drosophila, three beta-adrenergic-like octopamine receptors (Octβ1R, Octβ2R, and Octβ3R) have been identified. This study investigated the expression of three OctβR genes in embryos, larvae, and adults. These OctβRs showed distinct expression patterns in the central nervous system (CNS) throughout development, and Octβ3R expression was evident in an endocrine organ, the ring gland, in larvae. In larvae, Octβ1R, Octβ2R, and Octβ3R were expressed in salivary glands and imaginal discs, Octβ2R and Octβ3R in midgut, and Octβ3R in gonads. In adult, besides in the CNS, each OctβR was strongly expressed in ovary and testis. These findings provide a basis for understanding the mechanisms by which OctβRs mediate multiple diverse octopaminergic functions during development (Ohhara, 2012).

Identification and characterization of a novel family of Drosophila beta-adrenergic-like octopamine G-protein coupled receptors

Insect octopamine receptors carry out many functional roles traditionally associated with vertebrate adrenergic receptors. These include control of carbohydrate metabolism, modulation of muscular tension, modulation of sensory inputs and modulation of memory and learning. The activation of octopamine receptors mediating many of these actions leads to increases in the levels of cyclic AMP. However, to date none of the insect octopamine receptors that have been cloned have been convincingly shown to be capable of directly mediating selective and significant increases in cyclic AMP levels. This paper reports on the identification and characterization of a novel, neuronally expressed family of three Drosophila G-protein coupled receptors that are selectively coupled to increases in intracellular cyclic AMP levels by octopamine. This group of receptors, DmOct β1R (CG6919), DmOct β2R (CG6989) and DmOct β3R (CG7078) shows homology to vertebrate beta-adrenergic receptors. When expressed in Chinese hamster ovary cells all three receptors show a strong preference for octopamine over tyramine for the accumulation of cyclic AMP but show unique pharmacological profiles when tested with a range of synthetic agonists and antagonists. Thus, the pharmacological profile of individual insect tissue responses to octopamine might vary with the combination and the degree of expression of the individual octopamine receptors present (Maqueira, 2005).

Search PubMed for articles about Drosophila Octbeta3R

Branch, A., Zhang, Y. and Shen, P. (2017). Genetic and neurobiological analyses of the noradrenergic-like system in vulnerability to sugar overconsumption using a Drosophila model. Sci Rep 7(1): 17642. PubMed ID: 29247240

Maqueira, B., Chatwin, H. and Evans, P. D. (2005). Identification and characterization of a novel family of Drosophila beta-adrenergic-like octopamine G-protein coupled receptors. J Neurochem 94: 547-560. PubMed ID: 15998303

Ohhara, Y., Kayashima, Y., Hayashi, Y., Kobayashi, S. and Yamakawa-Kobayashi, K. (2012). Expression of beta-adrenergic-like octopamine receptors during Drosophila development. Zoolog Sci 29: 83-89. PubMed ID: 22303848

Ohhara, Y., Shimada-Niwa, Y., Niwa, R., Kayashima, Y., Hayashi, Y., Akagi, K., Ueda, H., Yamakawa-Kobayashi, K. and Kobayashi, S. (2015). Autocrine regulation of ecdysone synthesis by β3-octopamine receptor in the prothoracic gland is essential for Drosophila metamorphosis. Proc Natl Acad Sci USA [Epub ahead of print]. PubMed ID: 25605909

Zhang, T., Branch, A., Shen, P. (2013) Octopamine-mediated circuit mechanism underlying controlled appetite for palatable food in Drosophila. Proc Natl Acad Sci U S A. PubMed ID: 24003139

date revised: 25 April 2018

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