InteractiveFly: GeneBrief

Octopamine β 1 receptor & Octopamine β2 receptor: Biological Overview | References

BIOLOGICAL OVERVIEW

Adrenergic signaling has important roles in synaptic plasticity and metaplasticity. However, the underlying mechanisms of these functions remain poorly understood. This study investigated the role of octopamine, the invertebrate counterpart of adrenaline and noradrenaline, in synaptic and behavioral plasticity in Drosophila. It was found that an increase in locomotor speed induced by food deprivation is accompanied by an activity- and octopamine-dependent extension of octopaminergic arbors and that the formation and maintenance of these arbors required electrical activity. Growth of octopaminergic arbors is controlled by a cAMP- and CREB-dependent positive-feedback mechanism that required Octβ2R octopamine autoreceptors (a receptor located on presynaptic nerve cell membranes, serving as a part of a feedback loop in signal transduction, and sensitive only to those neurotransmitters or hormones that are released by the neuron in whose membrane the autoreceptor sits). Notably, this autoregulation was necessary for the locomotor response. In addition, octopamine neurons regulated the expansion of excitatory glutamatergic neuromuscular arbors through Octβ2Rs on glutamatergic motor neurons. These results provide a mechanism for global regulation of excitatory synapses, presumably to maintain synaptic and behavioral plasticity in a dynamic range (Koon, 2011).

The Drosophila larval neuromuscular junction (NMJ) is a powerful model system in which to investigate synaptic plasticity. Although glutamate is the primary excitatory neurotransmitter of the NMJ, larval NMJs are also innervated by octopaminergic motor neurons. Larval NMJs show several forms of synaptic plasticity, such as continuous expansion during larval development to offset a massive increase in muscle size, as part of a homeostatic mechanism to maintain synaptic efficacy. This process depends on signaling mechanisms such as the bone morphogenetic protein (BMP) and Wnt pathways. Larval NMJs can also respond to changes in the environment such as food availability by rapid increases in synapse strength. In addition, genetic and physiological manipulations that increase presynaptic activity promote synaptic expansion at NMJs. To determine the relevance of octopaminergic innervation of body-wall muscles, octopaminergic terminals were examined during larval foraging behavior. Type II arbors responded to food deprivation by extending new endings. This effect depended on both activity levels and octopamine. Electrical activity at octopaminergic neurons was essential for initial and continued type II innervation of muscles. A cAMP and CREB-dependent autoregulatory positive feedback mechanism was discovered that regulates the size of type II endings through the activation of Octβ2R autoreceptors. Type II innervation also regulates the plasticity of glutamatergic type I motor neurons through Octβ2Rs expressed in these neurons. Both the autocrine and paracrine mechanisms were required for the adaptive response to starvation (Koon, 2011).

Larval NMJs respond to acute changes in presynaptic activity by modifications in synaptic structure (Ataman, 2008). However, the physiological conditions under which this mechanism is used by the intact organism are unknown. Larval foraging behavior is enhanced by food deprivation, which leads to long-lasting enhancement of evoked glutamate release from excitatory type I NMJs (Steinert, 2006). However, no gross changes in the structure of these endings have been observed (Steinert, 2006). Most body-wall muscles are co-innervated by at least one additional class of motor neuron, the octopaminergic type II motor neuron (Monastirioti, 1995). Octopamine signaling has been implicated in appetitive behaviors and locomotion. Therefore, to determine whether type II arbors changed structure during starvation, a physiological stimulus that increases locomotor activity, these arbors were labelled by expressing mCD8-GFP using a tyrosine decarboxylase-2 (Tdc2) promoter fused to Gal4 (Tdc2-Gal4). NMJs were imaged in intact early third-instar larvae live through the cuticle, deprived the larvae of food for 2 h and then imaged the same NMJs again (Koon, 2011).

Food-deprived wild-type larvae showed a significant increase in locomotor speed compared to fed controls. Notably, type II endings in starved larvae showed dynamic filopodia-like extensions (synaptopods) that extended and retracted with a time course of minutes. Although synaptopods weree seen before food deprivation ('natural synaptopods'), the number of synaptopods was significantly increased upon starvation. Thus, changes in locomotor activity were accompanied by structural changes at type II endings (Koon, 2011).

Whether type II endings were necessary for behavior was investigated. Octopaminergic neurons were eliminated by expressing the cell-death protein head involution defective (Hid), which substantially reduced locomotor speed and the starvation response. A similar result has been observed in tyrosine beta-hydroxylase (tbh^nMI8) and tdc2^R054 mutants, which cannot synthesize octopamine. The defects in tbh mutants were specific, as they were rescued by expressing a TBH transgene in octopaminergic neurons. Thus, the increase in locomotion elicited by food deprivation results in structural changes in octopaminergic endings, and octopamine innervation is necessary for this behavior (Koon, 2011).

This study shows that octopamine regulates behavioral and synaptic plasticity through an autoregulatory mechanism that promotes the growth of type II innervation and in turn the expansion of excitatory glutamatergic arbors. This process seems to be associated with physiological stimuli that lead to increased locomotion. It is proposed that food deprivation elicits the release of octopamine by type II terminals. Octopamine binds to Octβ2R receptors and thereby increases cAMP, which activates CREB-dependent regulation of transcription and leads to new type II synaptic growth. This autoregulatory mechanism might control the amount of octopamine released by type II arbors. In turn, octopamine release stimulates the growth of type I arbors through Octβ2R at type I motor neurons. This mechanism would regulate, in a global fashion, excitatory transmission at the NMJ (Koon, 2011).

Increases in larval locomotion, type II motor neuron activity or exogenous octopamine resulted in the extension of synaptopods. With the demonstration that synaptopod extension constitutes a mechanism for the formation of type II boutons, these results suggest that the above events control the growth of octopaminergic endings in an acute manner. Analysis of mutations in octopamine receptors and components of the cAMP cascade revealed an autoregulatory mechanism that controls this growth. First, expression of Octβ2R in type II motor neurons was required for type II synaptic growth. Second, altering cAMP levels by mutations in dnc or rut modified this response in a manner consistent with positive regulation by cAMP. This regulation was cell autonomous in octopaminergic motor neurons, as the defects in synaptopod formation and type II synaptic growth were also elicited or rescued by transgene expression in octopaminergic motor neurons alone, in a chronic or acute manner. The presence of auto-octopamine receptors had been suggested in locusts (Howell, 1998), although the identity of the proposed autoreceptor was not known. However, it was proposed that the locust octopamine autoreceptors served to inhibit octopamine release. By contrast, the current experiments are consistent with a positive feedback mechanism that enhances synaptic growth. Autoregulatory mechanisms that control the amount of neuromodulator release have been previously demonstrated for neuromodulators such as dopamine (Koon, 2011).

As in other forms of synaptic plasticity, including late LTP and long-term memory, the autoregulatory mechanism required the function of CREB and new protein synthesis. This finding underscores the universality of mechanisms by which the nervous system modifies the efficacy of connections in a long-lasting manner. Octopamine receptor activation leading to CREB signaling has also been demonstrated in Caenorhabditis elegans (Suo, 2006; Koon, 2011 and references therein).

These studies showed that this pathway regulates the structure of octopaminergic arbors in an autoregulatory fashion, and that this influences the growth of type I excitatory arbors. The presence of a positive feedback that controls the growth of modulatory inputs in an acute manner provides a mechanism by which animal experience can modify circuitry and thus by which animals can adapt to a changing environment (Koon, 2011).

Activity is absolutely required for innervation of body-wall muscles by type II arbors, as reduced activity perturbs type II synaptogenesis. This is in contrast to the widely held view that although activity is important for the refinement of connections, it is not required for initial synaptogenesis. Part of this view arises from the examination of arbors that mediate classical neurotransmission. By contrast, the dependence of modulatory terminal growth on activity has been less studied. These studies provide compelling evidence that octopamine has an influence on bouton outgrowth in octopaminergic type II and type I motor neurons. Studies of type I bouton outgrowth have identified local factors that influence the development of pre- or postsynaptic compartments, including Wnts and BMPs. It is suggested that octopamine release by type II arbors might mediate a more global regulation of outgrowth (Koon, 2011).

At the Drosophila larval NMJ, glutamatergic type Ib motor neurons innervate each muscle in an approximately 1:1 manner. In addition, two glutamatergic type Is motor neurons innervate the entire ventral or dorsal muscle field within each hemisegment. By contrast, the three octopaminergic neurons per segment innervate most of the body-wall muscles in a bilateral fashion (Monastirioti, 1995). The layout of this innervation suggests that type II synapses might establish global regulation of the plasticity of type I arbors. This might serve as a mechanism for setting excitability levels in the entire body wall, and thereby keep synaptic function in a dynamic range. Similarly, studies in mammalian systems have shown that adrenergic signaling can affect plasticity at glutamatergic synapses, either through changes in ionotropic GluR localization (Hu, 2007) or through regulation of metabotropic GluR, which affects the ability of a synapse to become potentiated depending on its history (Kuzmiski, 2009). Octopamine might regulate the ability of type I NMJs to trigger muscle contraction by long-term regulation of type I synaptic growth (Koon, 2011).

Two previous studies at the Drosophila larval NMJ have shown that octopamine enhances synaptic transmission (Nagaya, 2002; Kutsukake, 2000). However, another study reported that octopamine might inhibit glutamatergic transmission in first-instar larvae (Nishikawa, 1999). The current studies suggest that blocking activity or interfering with octopamine signaling in type II neurons leads to a decrease in type I synaptic outgrowth, consistent with the idea that octopamine release is a positive regulator of type I transmission. It is suggested that in the short term, octopamine enhances synaptic strength, as observed in electrophysiology experiments, leading to the observed increase in crawling behavior after starvation. This would be consistent with studies showing that increases in locomotor speed induced by food deprivation led to an enhancement of synaptic efficacy (Koon, 2011).

Octopamine is a potent modulator of invertebrate behavior and is secreted during starvation in invertebrates. Nevertheless, its function at the synaptic level is poorly understood. This study shows that octopamine can influence synapses at the structural level through the activation of Octβ2R autoreceptors in octopamine neurons and through the presence of these receptors in type I motor neurons (Koon, 2011).

An important question is whether octopamine is simply involved in locomotion and the lack of starvation response in mutants that cannot synthesize octopamine is an indirect effect of defective locomotion. It is not possible to answer this question in tbh mutants, as basal locomotion is reduced in these mutants. However, the current experiments reveal conditions in which changes in basal activity can be genetically separated from changes in the starvation response. One such case is rut mutants, which have normal locomotion but lack the starvation response. This effect seems to be due to the function of Rut in octopamine neurons, as the defective starvation response is completely rescued by expressing a Rut transgene in octopamine neurons. A second, albeit less clear observation regards octβ2R mutants. Although baseline locomotion was much less altered in these mutants than in tbh mutants, these animals still could not mount a starvation response. Thus, it is likely that octopamine neurons are involved not only in locomotion, but also in the response to starvation (Koon, 2011).

Octopamine is also required for appetitive memory in adult fruit flies (Schwaerzel, 2003). Notably, the appetitive memory procedure requires starvation before the assay, and tbh mutants cannot learn in this procedure. Octopamine has been proposed to mediate the reinforcing effects of sugar in appetitive memory formation. The current studies raise the possibility that this mechanism might involve structural changes at synaptic sites (Koon, 2011).

Although these studies focused on structural changes at type II NMJs, many of the manipulations affected all octopamine neurons, as Tdc2-Gal4 drives Gal4 in all octopaminergic neurons. Thus, these studies cannot rule out an influence from other octopaminergic neurons, besides motor neurons, in the changes observed and in the behavior. However, the finding that the manipulations resulted in specific changes in type II NMJ terminals and that octopamine modulates synaptic strength at the NMJ argues that at least some of the effects are likely to be due to the peripheral octopamine innervation (Koon, 2011).

In summary, these studies reveal important mechanisms by which activity regulates the ability of motor neurons to scale the release of regulatory signals, which is important for the adaptation of the organism to the environment. In addition, they show a mechanism by which excitatory synapses are regulated in a global manner, presumably to maintain synaptic plasticity in a dynamic range (Koon, 2011).

Inhibitory control of synaptic and behavioral plasticity by octopaminergic signaling

Adrenergic receptors and their ligands are important regulators of synaptic plasticity and metaplasticity, but the exact mechanisms underlying their action are still poorly understood. Octopamine, the invertebrate homolog of mammalian adrenaline or noradrenaline, plays important roles in modulating behavior and synaptic functions. Previous work (Koon, 2011) uncovered an octopaminergic positive-feedback mechanism to regulate structural synaptic plasticity during development and in response to starvation. Under this mechanism, activation of Octβ2R autoreceptors by octopamine at octopaminergic neurons initiated a cAMP-dependent cascade that stimulated the development of new synaptic boutons at the Drosophila larval neuromuscular junction (NMJ). However, the regulatory mechanisms that served to brake such positive feedback were not known. This study reports the presence of an alternative octopamine autoreceptor, Octβ1R, with antagonistic functions on synaptic growth. Mutations in octβ1r result in the overgrowth of both glutamatergic and octopaminergic NMJs, suggesting that Octβ1R is a negative regulator of synaptic expansion. As does Octβ2R, Octβ1R functions in a cell-autonomous manner at presynaptic motorneurons. However, unlike Octβ2R, which activates a cAMP pathway, Octβ1R likely inhibits cAMP production through inhibitory Goα. Despite its inhibitory role, Octβ1R is required for acute changes in synaptic structure in response to octopamine and for starvation-induced increase in locomotor speed. These results demonstrate the dual action of octopamine on synaptic growth and behavioral plasticity, and highlight the important role of inhibitory influences for normal responses to physiological stimuli (Koon, 2012).

Adrenaline/noradrenaline and their receptors have emerged as important modulators of synaptic plasticity, metaplasticity, and behavior in the mammalian brain (Murchison, 2004; Hu, 2007; Kuzmiski, 2009). However, the mechanisms underlying this regulation of synaptic structure are not known (Koon, 2012).

In insects, adrenergic signaling is accomplished through octopamine and octopamine receptors (Balfanz, 2005) and is a powerful modulator of behaviors such as appetitive behavior and aggression. It also regulates synaptic function and synaptic structure (Koon, 2011; Koon, 2012).

A previous study has demonstrated that at the Drosophila larval neuromuscular junction (NMJ) octopamine regulates the expansion of both modulatory and excitatory nerve terminals (Koon, 2011). Larval NMJs are innervated by glutamatergic, octopaminergic, and peptidergic motorneurons. Of these, glutamatergic nerve terminals provide classical excitatory transmission, while octopaminergic nerve endings support global modulation of excitability and synaptic growth. Larval NMJs are continuously expanding to compensate for muscle cell growth and respond to acute changes in activity by extending new synaptic boutons. By binding to the octopamine autoreceptor Octβ2R, octopamine activates a cAMP second messenger pathway that leads to CREB activation and transcription, which in turn promotes the extension of new octopaminergic nerve endings (Koon, 2011). This positive-feedback mechanism was required for an increase in locomotor activity in response to starvation. In addition, this mechanism positively regulated the growth of glutamatergic nerve endings through Octβ2R receptors present in glutamatergic neurons. An important question regards the mechanisms that serve to brake such positive feedback (Koon, 2012).

This study demonstrates the presence of a second octopamine receptor, Octβ1R, which serves as such a brake. Octβ1R is also an autoreceptor in octopaminergic neurons that serves to inhibit synaptic growth. This inhibitory influence is excerpted through the activation of the inhibitory G-protein subunit, Goα, and thus by limiting cAMP production. Like Octβ2R receptors, Octβ1R receptors are also present at excitatory glutamatergic endings. Thus, octopamine release induces a dual excitatory (through Octβ2R) and inhibitory (through Octβ1R) function on the growth of both octopaminergic and glutamatergic endings. The presence of both the excitatory and inhibitory receptors is required for normal structural plasticity at octopaminergic terminals and for normal responses to starvation, as obliterating Octβ1R (this study) or Octβ2R (Koon, 2011) prevents the acute growth of octopaminergic ending in response to octopamine and the increase in locomotor speed in response to starvation. Thus, this study highlights the requirement of both excitatory and inhibitory influences for normal synaptic and behavioral plasticity (Koon, 2012).

The previous study demonstrated that octopamine regulates synaptic and behavioral plasticity through an autoregulatory positive-feedback mechanism involving Octβ2R, which promotes both type I and type II outgrowth (Koon, 2011). This study has now identified an octopamine receptor, Octβ1R, which antagonizes the function of Octβ2R. It is proposed that Octβ1R may serve as a brake for the positive feedback induced by Octβ2R. Octβ1R receptors inhibit the cAMP pathway via the inhibitory G-protein Goα, as loss of Octβ1R or Goα function results in synaptic overgrowth of type I and type II endings in an octopamine cell-autonomous manner, and as octβ1r and goα interact genetically. Notably, defective Octβ1R signaling appears to saturate cAMP levels, occluding the function of Octβ2R. Thus, the loss of Octβ1R function results in insensitivity to octopamine stimulation. In turn, this abolishes starvation-induced behavioral changes that require Octβ2R signaling. While this study centered primarily on Octβ1R function at octopaminergic NMJ terminals, it is important to emphasize that octopamine neurons are also present in the larval brain. Thus, with current tools it is not possible to discern whether the defects are exclusively due to the function of octopaminergic motorneurons, or whether other central octopaminergic neurons contribute to these effects. While the phenotypes on NMJ development are most parsimoniously explained by a local function at NMJ terminals, it is likely that the behavioral effects are more complex, also involving important contribution from brain octopaminergic neurons (Koon, 2012).

At the Drosophila larval NMJ, three type II motorneurons innervate most of the body wall muscles in each segment (Koon, 2011). This layout suggests that octopamine is likely to globally regulate plasticity, by tuning the excitability levels of multiple excitatory synapses on the body wall muscles. Together, the observations in the previous study (Koon, 2011) and this investigation identify the presence of excitatory and inhibitory octopamine receptors that are coexpressed in the same cells. This suggests that global regulation of synapses and behavior by octopamine can be tipped toward excitation or inhibition depending on receptor expression levels, affinity of the receptors for octopamine, and availability of these receptors for binding octopamine on the target cells. This dual mode of controlling excitability likely provides enhanced flexibility, allowing a broader level of control over synaptic functions (Koon, 2012).

An important question is how can Octβ1R and Octβ2R regulate development of innervation and behavior given that they are activated by the same ligand, are localized in the same cells, and their functions are antagonistic. Several alternatives can be proposed. Octβ1R and Octβ2R might have different affinities for octopamine binding. Thus, different levels of octopamine release could differentially activate the receptors. For instance, if Octβ1R receptors have higher affinity for octopamine, and octopamine is normally released at low levels, a stable degree of innervation could be maintained by continuous inhibition of synaptic growth-promoting signals. High levels of octopamine release, as would occur during starvation, would then activate the lower affinity Octβ2R, eliciting synaptic growth. Precedence for this type of regulation has been obtained in honeybees and olive fruit flies, where low concentrations of octopamine are inhibitory while high concentrations are excitatory to cardiac contraction (Papaefthimiou, 2011; Koon, 2012).

An alternative possibility is based on the well known internalization of GPCRs upon ligand binding. It is possible that such a mechanism would maintain an appropriate ratio of Octβ1R and Octβ2R at the cell surface, actively keeping or removing octopamine receptor-mediated excitation or inhibition, depending on physiological states. A third alternative is that receptors could be posttranslationally modified upon ligand binding, which might also affect their downstream functions. For example, dimerization of β2-adrenergic receptors can inhibit its adenylate cyclase-activating activity and phosphorylation of β1-adrenergic receptor by PKA reduces its affinity for Gsα and increases its affinity for Giα/oα. Last, Octβ1R and Octβ2R receptors could be spatially separated in neurons, with one receptor being closer and the other distant to sites of octopamine release. In this scenario, the receptors would likely be exposed to different octopamine concentration (Koon, 2012).

Simultaneous expression of excitatory and inhibitory GPCRs in the same neuron has been reported previously. For instance, mammalian dopamine receptors can couple to both stimulatory and inhibitory G-proteins, with the D₁ receptor-like family being coupled to Gsα and the D₂-like family being coupled to Giα/oα (Koon, 2012).

Previous studies have investigated the effect of octopamine on synaptic transmission at the Drosophila first-instar and third-instar larval NMJ. While the studies at the third-instar larval NMJ demonstrated an excitatory effect of octopamine in neurotransmission, the study on the first-instar larval stage substantiated an inhibitory effect. A recent study now provides a potential explanation for such discrepancy between the responses to octopamine at the two larval stages (Ohhara, 2012). In particular, it was found that that Octβ1R is expressed at high levels in first instar and at low levels in third instar. In contrast, Octβ2R is expressed at low levels in first instar and at high levels in third instar (Ohhara, 2012). The current studies demonstrating an inhibitory role for Octβ1R (this study) and an excitatory role for Octβ2R (Koon, 2011) are in agreement with the idea that octopamine may play an inhibitory role during first instar, but an excitatory role during third instar (Koon, 2012).

Octopamine receptors have been shown to elicit intracellular Ca²⁺ and/or cAMP increase (Han, 1998; Balfanz, 2005). OAMB, the only α-adrenergic-like receptor in Drosophila, has been implicated to function via Ca²⁺ signaling in the Drosophila oviduct (Lee, 2009). However, OAMB is expressed in the oviduct epithelium, and not in the oviduct muscle cells (Lee, 2009). Given that octopamine induces relaxation of oviduct muscles, the presence of an alternative, inhibitory octopamine receptor in oviduct muscles was proposed (Lee, 2009). The identification of Octβ1R receptor as an inhibitory receptor raises the possibility that this is the inhibitory receptor in the oviduct (Koon, 2012).

In apparent contradiction to these findings, a previous study has shown that Octβ1R (also known as OA2) is capable of increasing cAMP (Balfanz, 2005). In that study, HEK293 cells transfected with Octβ1R were exposed to different octopamine concentrations, which resulted in an increase in cAMP levels (Balfanz, 2005). A potential explanation for the disparate results is that GPCR overexpression might alter its coupling to downstream pathways. For instance, mammalian β2-adrenergic receptors are known to couple to both Gsα and Giα/oα proteins. However, overexpression of β2-adrenergic receptors constitutively couples the receptor to Gsα and not to Giα or Goα. Furthermore, analysis of its binding specificity through immunoprecipitation shows that, when the receptor was overexpressed in transgenic mice, it coprecipitated with Gsα but not with Giα/oα in the absence of agonist. An additional explanation is that human embryonic kidney HEK293 cells are unlikely to express the same transduction pathways as endogenous Drosophila cells. Indeed, a recent study showed that HEK293 cells express virtually no Goα, which could also explain the lack of inhibitory response of overexpressed Octβ1R in this cell line (Koon, 2012).

Goα is expressed in the nervous system of Drosophila and shows a marked increase in levels during the development of axonal tracts. Goα levels are altered in memory mutants including dunce and rutabaga, and Goα is necessary for associative learning. PTX overexpression in mushroom bodies of adult Drosophila severely disrupts memory, suggesting a role of Goα in synaptic plasticity. However, homozygous goα mutants are lethal due to defective development of the heart preventing the use of null mutants in studies of the NMJ or the adult brain. Moreover, overexpression of inhibitory G-proteins is known to sequester available Gβ and Gγ subunits, resulting in unspecific downregulation of other G-protein signaling. Thus, there are significant problems associated with the use of an overexpression approach to study Goα function. Fortunately, the availability of PTX and multiple Goα-RNAi strains allowed downregulation of Goα function in a cell-specific manner to examine synaptic development at the NMJ, which was found to phenocopy defects observed at the NMJ of octβ1r mutants. The presence of genetic interactions between the octβ1r and goα genes further support the notion that the two proteins act in the same signaling pathway to inhibit synaptic growth. These results provide strong evidence for the involvement of Goα in synaptic plasticity at the NMJ (Koon, 2012).

In summary, these studies reveal that octopamine acts both as an inhibitory and excitatory transmitter to regulate synaptic growth and behavior. Thus, the inhibitory function of octopamine in global synaptic growth is as crucial as its excitatory function in maintaining plasticity in a dynamic range (Koon, 2012).

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. This study shows, 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 alpha-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 beta-adrenergic-like OCTbeta2R 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 (Burke, 2012).

Fruitfly octopamine is synthesized from tyrosine via two steps catalysed by tyrosine decarboxylase (TDC) and tyramine β-hydroxylase (Tbh). The Tdc2 gene encodes the neuronal TDC and a Tdc2-GAL4 construct (where GAL4 is cloned downstream of a Tdc2 promoter fragment) can be used to label and manipulate many of the octopamine neurons. Although Tbh mutant Drosophila that lack octopamine cannot form appetitive memory (Schwaerzel, 2003), the precise role of octopamine release is currently unknown (Burke, 2012).

This study tested whether octopamine neurons were required for appetitive olfactory conditioning with sucrose reinforcement by blocking them throughout the experiment using Tdc2-GAL4-driven UAS-shibire^ts1 (UAS-shi^ts1, where the shi^ts1 sequence is cloned downstream of the upstream activation sequence (UAS). The UAS-shi^ts1 transgene allows temporary blockade of synaptic transmission from specific neurons by shifting flies from the permissive temperature <25°C to the restrictive >29°C. Tdc2-GAL4;UAS-shi^ts1 were assayed flies in parallel with GAL4 driver, UAS-shi^ts1 transgene and wild-type flies for comparison. All flies were incubated at 31°C to disrupt output from octopamine neurons for 30min before being trained and tested for 3h appetitive memory at 31°C. Surprisingly, no defects were apparent (Burke, 2012).

Sweet taste and nutrient value both contribute to appetitive memory reinforcement in Drosophila. It was reasoned that octopamine blockade might lack consequence if octopamine only represents sweet taste and nutrient value provides sufficient reinforcement. Therefore Tdc2 neurons were blocked while training flies with arabinose, a sweet but non-nutritious sugar. All flies were trained and tested for 3min memory at 31°C. In this case, memory of Tdc2-GAL4;UAS-shi^ts1 flies was significantly impaired compared to all control groups. Importantly, no significant differences were apparent between groups trained and tested at 25°C. To further challenge a nutrient bypass model octopamine neurons were blocked while flies were conditioned with arabinose supplemented with nutritious sorbitol, similar to blocking octopamine neurons in flies conditioned with sweet and nutritious sucrose. These data are consistent with octopamine only conveying the reinforcing effects of sweet taste and with nutrient value being sufficient for appetitive learning (Burke, 2012).

To determine whether octopamine provides instructive reinforcement flies were conditioned with odour presentation paired with artificial octopamine neuron activation, achieved by expressing UAS-dTrpA1 with Tdc2-GAL4. dTrpA1 (also known as TrpA1) encodes a transient receptor potential (TRP) channel that conducts Ca²⁺ and depolarizes neurons when flies are exposed to temperature >25°C. Ad-libitum-fed wild-type, Tdc2-GAL4, UAS-dTrpA1 and Tdc2-GAL4;UAS-dTrpA1 flies were conditioned by presenting an odour with activating 31°C, and immediately tested for memory. Tdc2-GAL4;UAS-dTrpA1 flies exhibited robust appetitive memory that was statistically different from all other groups. Significant memory remained at 30min in satiated flies but was statistically indistinguishable from all other groups at 3h, even in hungry flies. Therefore appetitive memory implanted with octopamine neuron activation is short-lived. Tdc2-GAL4 is expressed in neurons that contain and could release tyramine, either alone or together with octopamine. To confirm that artificial learning requires octopamine stimulated Tdc2 neurons were stimulated in Tbh mutant flies that cannot synthesize octopamine from tyramine. No learning was observed, indicating that octopamine release is required for artificial learning (see Octopamine neuron stimulation can replace sugar presentation during conditioning to form short-term appetitive memory) (Burke, 2012).

Although octopamine neuron innervation of the mushroom body (MB) is relatively sparse in the γ lobe, heel and calyx, previous work suggests that MB neurons are probably the eventual destination of appetitive reinforcement signals. Therefore the NP7088, 0665 and 0891-GAL4 lines were used to investigate the role of the four individual classes of octopamine (OA) neurons that innervate the MB. NP7088 neurons visualized using UAS-mCD8::GFP (that is, expressing the green fluorescent protein (GFP)) broadly overlap with Tdc2-GAL4 neurons in the brain, but NP7088-GAL4 does not label OA-VPM5 neurons . 0665-GAL4 driven GFP expression is even more restricted and labels the OA-VPM3 and OA-VPM4 neurons. Finally, 0891-GAL4 only labels OA-VPM4 (Burke, 2012).

Activating these subpopulations of MB-innervating octopamine neurons during odour presentation did not form appetitive memory. Similarly, blocking them (NP7088, 0665 or 0891-GAL4) using UAS-shi^ts1 did not significantly impair arabinose-reinforced memory. Importantly, these data indicate that the fly equivalent of the bee VUMmx1 neuron, OA-VUMa, and the other MB-innervating neurons covered by these drivers, are neither sufficient nor essential for conditioned olfactory approach behaviour in flies. Instead, the data indicate that either the calyx-innervating OA-VPM5 neurons are critical, or a more distributed octopamine signal involving other non-MB-innervating octopamine neurons is required for appetitive reinforcement (Burke, 2012).

One study implicated the DopR dopamine receptor in appetitive memory. Flies with the dumb¹ mutation have impaired appetitive memory that can be restored by expressing DopR in the MB. Therefore this study tested whether memory formation with octopamine neuron activation required DopR. No significant memory was observed in any group carrying dumb¹. Therefore a functional dopamine system is required to form appetitive memory with octopamine, suggesting that dopamine is downstream of octopamine in appetitive memory processes (Burke, 2012).

A recent study implicated dopamine neurons in the PAM (paired anterior medial) cluster in appetitive reinforcement (Liu, 2012). 0273-GAL4 and 0104-GAL4 lines in the InSITE collection were identified that drive UAS-mCD8::GFP in subsets of PAM dopamine neurons that innervate the MB. Co-labelling brains with UAS-mCD8::GFP and anti-tyrosine hydroxylase (TH) antibody revealed that 0273-GAL4 is expressed in all of the ~130 dopamine neurons in the PAM cluster, whereas 0104-GAL4 labels a subset of 40 PAM dopamine neurons. Importantly, neither line labels dopamine neurons in the paired posterior lateral 1 (PPL1) cluster that convey negative value (Burke, 2012).

Whether 0104-GAL4 and 0273-GAL4 PAM neurons could provide appetitive reinforcement was tested by activating them with UAS-dTrpA1 while presenting an odour in satiated flies. Both 0104-GAL4;UAS-dTrpA1 and 0273-GAL4;UAS-dTrpA1 flies exhibited robust appetitive memory that was statistically different from all control flies and far greater than scores observed with a similar stimulation of octopamine neurons (Burke, 2012).

Because 0104-GAL4 more precisely labels reinforcing PAM dopamine neurons than 0273-GAL4, 0104-GAL4 was used with UAS-shi^ts1 to test whether output from PAM dopamine neurons was required for appetitive learning with sugar reinforcement. The 0104-GAL4;UAS-shi^ts1 flies were tested in parallel with GAL4 driver, UAS-shi^ts1 transgene and wild-type flies for comparison. Blocking 0104-GAL4 neurons abolished memory in arabinose-conditioned flies. The initial memory performance of sucrose-conditioned flies was also significantly impaired. Moreover, sucrose-conditioned 24h memory was abolished if 0104-GAL4 neurons were only blocked during training. Importantly, training and testing the flies at the permissive temperature did not impair performance. Therefore PAM dopamine neurons, like octopamine neurons, are critical for conditioning with arabinose but, unlike octopamine neurons, they also contribute towards the reinforcing effects of nutritious sucrose (Burke, 2012).

Tbh mutant flies that lack octopamine were artificially conditioned to investigate further whether dopamine reinforcement is downstream of octopamine. Appetitive memory formed in Tbh flies with 0104-GAL4;UAS-dTrpA1 or 0273-GAL4;UAS-dTrpA1 was statistically indistinguishable from that formed in the wild-type background, confirming that dopamine-mediated reinforcement is downstream, and can function independently, of octopamine (Burke, 2012).

To investigate a plausible direct link between octopamine and dopamine neurons tests were performed to see whether octopamine neuron activation could form memory in octopamine receptor mutant flies. Artificial learning worked effectively in satiated octβ1R (also known as oa2) mutant flies, but was impaired in hungry oamb mutant flies, suggesting a key role for OAMB in reinforcement. To determine whether Oamb is required in PAM dopamine neurons UAS-Oamb^RNAi was expressed with 0104-GAL4, and flies were conditioned with arabinose. The memory of 0104-GAL4;UAS-Oamb^RNAi flies was significantly different to that of both control groups. The same flies were also tested conditioned with sucrose. Consistent with previous experiments with octopamine manipulation, no effect was observed with this nutritious sugar. Therefore, the OAMB receptor is required in PAM dopamine neurons for octopamine-dependent memory (Burke, 2012).

OAMB couples to calcium release from intracellular stores. Therefore GCaMP3.0 (Tian, 2009) was tested in PAM dopamine neurons with 0104-GAL4, and intracellular Ca²⁺ responses evoked by application of exogenous octopamine were tested. Octopamine application drove a significant increase in Ca²⁺ signal in PAM dopamine neurons that was abolished by pre-exposing the brain to the octopamine receptor antagonist mianserin. Therefore behavioural, anatomical and physiological data are consistent with octopamine-dependent reinforcement involving OAMB-directed modulation of PAM dopamine neurons (Burke, 2012).

Studies in octβ2R (Maqueira, 2005) mutant flies revealed a more nuanced picture for octopamine-mediated reinforcement. Artificial learning with octopamine neuron activation was impaired in satiated octβ2R/+ heterozygous flies), but was restored in octβ2R/+ flies by food-deprivation. These data suggest that octopamine also integrates with systems that are responsive to hunger to provide instructive reinforcement. Such a role for octopamine is also highlighted by the observation of memory performance in all previous experiments using satiated flies (Burke, 2012).

Previous work demonstrated that fly neuropeptide F (dNPF) modulates the MB–heel-innervating MB-MP1 dopamine neurons to limit retrieval of appetitive memory performance to hungry flies (Krashes, 2009). Artificial learning with octopamine worked effectively in dNPF receptor mutant flies, indicating that octopamine functions independently of dNPF. Therefore a role for Octβ2R was tested in MB-MP1 dopamine neurons. A new Tdc2-LexA was used to simultaneously express lexAop-dTrpA1 in octopamine neurons and UAS-Octβ2R^RNAi in MB-MP1 neurons using c061-GAL4;MBGAL80. Hungry Tdc2-LexA;lexAop-dTrpA1 flies formed robust appetitive memory. However, Tdc2-LexA;lexAop-dTrpA1 flies that also carried c061;MBGAL80;UAS-Octβ2R^RNAi transgenes to knockdown Octβ2R expression in MB-MP1 neurons did not display memory. The role of MB-MP1 neurons in octopamine-mediated reinforcement was tested independently by simultaneously stimulating octopamine neurons while disrupting output from MB-MP1 neurons with UAS-shi^ts1. Flies in which MB-MP1 neurons were simultaneously blocked during artificial conditioning showed no significant memory. Because MB-MP1 neurons can provide aversive reinforcement if artificially engaged during odour presentation, they probably provide a negative influence to the system. Therefore, the data indicate that octopamine-dependent appetitive reinforcement requires OCTβ2R modulation of negative dopamine signals from MB-MP1 neurons in addition to OAMB signalling in positive PAM dopamine neurons (Burke, 2012).

The 0104-GAL4 dopamine neurons have presynaptic terminals in the tip of MB β′ and γ lobes and presumed dendrites in the anterior medial protocerebrum (ampr). Green fluorescent protein reconstituted across synaptic partners (GRASP) was used to investigate plausible sites of synaptic contact between octopamine neurons and PAM and MB-MP1 dopamine neurons. lexAop-mCD4::spGFP11 was expressed with Tdc2-lexA and UAS-mCD4::spGFP1-10 with 0104-GAL4 or c061-GAL4;MBGAL80. Both of these combinations revealed strong GFP labelling in the ampr. In addition, MB-MP1 dopamine neuron:octopamine GRASP labelled the MB–heel region. The best candidates to bridge these two regions are the OA-VPM4 neurons, which densely innervate the MB heel and γ lobes and the ampr. However, OA-VPM4 neurons are cleanly labelled in 0891-GAL4 and NP7088 GAL4 lines, all of which were insufficient for appetitive conditioning. The rest of the ampr-innervating neurons, OA-VUMa6, OA-VUMa7, OA-VUMa8 and OA-VPM3, are also included in the NP7088 GAL4-labelled population. Finally, the MB-calyx-innervating OA-VPM5 neurons that are in Tdc2-GAL4 but not NP7088 do not have arborizations in the ampr or MB heel, so cannot provide direct modulation of PAM or MB-MP1 dopamine neurons. Therefore, reinforcing octopamine in the fly is provided by a distributed set of neurons, some of which have arborizations in the ampr where they modulate reinforcing PAM and MB-MP1 dopamine neurons. It is speculated that octopamine reinforcement may also require simultaneous regulation of other unidentified dopamine neurons, or involvement of additional parallel modes of octopamine action (Burke, 2012).

An octopamine-mushroom body circuit modulates the formation of anesthesia-resistant memory in Drosophila

Drosophila olfactory aversive conditioning produces two components of intermediate-term memory: anesthesia-sensitive memory (ASM) and anesthesia-resistant memory (ARM). Recently, the anterior paired lateral (APL) neuron innervating the whole mushroom body (MB) has been shown to modulate ASM via gap-junctional communication in olfactory conditioning. Octopamine (OA), an invertebrate analog of norepinephrine, is involved in appetitive conditioning, but its role in aversive memory remains uncertain. This study shows that chemical neurotransmission from the anterior paired lateral (APL) neuron, after conditioning but before testing, is necessary for aversive ARM formation. The APL neurons are tyramine, Tβh, and OA immunopositive. An adult-stage-specific RNAi knockdown of Tβh in the APL neurons or Octβ2R OA receptors in the MB α'β' Kenyon cells (KCs) impaired ARM. Importantly, an additive ARM deficit occurred when Tβh knockdown in the APL neurons was in the radish mutant flies or in the wild-type flies with inhibited serotonin synthesis. It is concluded that OA released from the APL neurons acts on α'β' KCs via Octβ2R receptor to modulate Drosophila ARM formation. Additive effects suggest that two parallel ARM pathways, serotoninergic DPM-αβ KCs and octopaminergic APL-α'β' KCs, exist in the MB (Wu, 2013).

The key finding of this study is that OA from the single APL neuron innervating the entire MB is required specifically for ARM formation in aversive olfactory conditioning in Drosophila. This conclusion is supported by five independent lines of evidence. First, blocking neurotransmission from APL neurons after training, but before testing, impaired ARM. Second, the APL neurons are tyramine, Tβh, and OA antibody immunopositive. Third, adult-stage-specific reduction of Tβh levels in the APL neurons, but not in dTdc2-GAL4 neurons that do not include the APL neurons, specifically abolishes ARM without affecting learning or ASM. Fourth, Octβ2R is expressed preferentially in the α'β' lobes, and adult-stage-specific reduction of Octβ2R expression in the α'β' KCs impaired ARM. Fifth, the additive memory impairments demonstrated in flies subjected to Tβh plus inx7 knockdowns and Tβh knockdown plus cold shock, but not inx7 knockdown plus cold shock, confirm that a single APL neuron modulates both ASM and ARM through gap-junctional communication and OA neurotransmission, respectively. Although it has been shown that the APL neurons are also GABAergic, the current results showed that OA is the primary neurotransmitter from the APL neurons involved in ARM formation because reduced GABA levels induced by Gad1^RNAi inhibition in the APL neurons did not affect 3 hr memory (Wu, 2013).

In Drosophila olfactory memories, OA and dopamine have been shown to act as appetitive and aversive US reinforcements, respectively. It is important to point out that the original claim that Tβh plays no role in aversive learning only examined 3 min memory, not 3 hr memory or ARM. It is not surprising to find that OA modulates ARM in aversive memory because dopamine has also been attributed to diverse memory roles, including a motivation switch for appetitive ITM and appetitive reinforcement. Intriguingly, dopamine negatively inhibits ITM formation, but OA positively modulates ARM formation (Wu, 2013).

Food deprivation in Drosophila larvae induces behavioral plasticity and the growth of octopaminergic arbors via Octβ2R-mediated cyclic AMP (cAMP) elevation in an autocrine fashion. This study showed that the APL neurons release OA acting on the Octβ2R-expressing α' β' KCs for ARM, instead of inducing autocrine regulation. Applying OA directly onto the adult brain results in an elevation of cAMP levels in the whole MB, and OA has been shown to upregulate protein kinase A (PKA) activity in the MBs. Intriguingly, ARM is enhanced by a decreased PKA activity and requires DUNCE-sensitive cAMP signals. It is speculated that APL-mediated activation of Octβ2R may lead to an intricate regulation of cAMP in the α' β' KCs for ARM formation. (Wu, 2013).

Although it has generally been assumed that, in a particular neuron, the same neurotransmitter is used at all synapses, exceptions continue to accumulate in both vertebrates and invertebrates. Scattered evidence suggests that co-release may be regulated at presynaptic vesicle filling and postsynaptic activation of receptors, but the physiologic significance remains poorly understood. This study reports that the APL neurons co-release GABA and OA. In the APL neurons, a reduced GABA level affects learning, but not ITM, whereas a reduced OA level has no effect on learning, but impairs ITM, suggesting that the two neurotransmitters are regulated in different ways in the same cell (Wu, 2013).

It has been proposed that the APL neurons might be the Drosophila equivalent of the honeybee GABAergic feedback neurons, receiving odor information from the MB lobes and releasing GABA inhibition to the MB calyx. This negative feedback loop for olfactory sparse coding has been supported by electrophysiological recording of the giant GABAergic neuron in locusts. However, the function of Drosophila APL neurons is complicated by the existence of functioning presynaptic processes in the MB lobes, mixed axon-dendrite distribution throughout the whole MB, and GABA/OA cotransmission (Wu, 2013).

Normal performance of ARM behavior requires serotonin from the DPM neurons acting on ab KCs via d5HT1A serotonin receptors and function of RADISH and BRUCHPILOT in the ab KCs. Surprisingly,the current results show that ARM formation also requires OA from the APL neurons acting on the α' β' KCs via Octβ2R OA receptors, suggesting the existence of two distinct anatomical circuits involved in ARM formation. However, it remains uncertain whether two branches of ARM occur in parallel because combination of various molecular disruptions (i.e., TβhRNAi and pCPA feeding/rsh¹ mutant) did not completely abolish ARM and partial disruption of one anatomical circuit will allow additive effects of another treatment even if they act on the same ARM. The hypothesis of the existence of two distinct forms of ARM is favored based on the following observations. First, neither d5HT1A^RNAi knockdown in α' β' KCs nor Octβ2R^RNAi knockdown in ab KCs affects ARM, suggesting that the two signaling pathways act separately in different KCs and do not affect each other in the same KCs. Second, each of the three ways of molecular disruption (i.e., Tβh^RNAi, pCPA feeding, and rsh¹ mutant) results in a similar degree of ARM impairment, but additive effect did not occur in rsh¹ mutant flies fed with pCPA and was evident when Tβh^RNAi treatment combines with either pCPA feeding or rsh¹ mutant. It's noteworthy that ARM is also affected by dopamine modulation because calcium oscillation within dopaminergic MB-MP1 and MB-MV1 neurons controls ARM and gates long-term memory, albeit a different view has been brought up. The target KCs of these dopaminergic neurons on ARM remain to be addressed (Wu, 2013).

Both the APL and DPM neurons are responsive to electric shock and multiple odorants, suggesting that they likely acquire olfactory associative information during learning for subsequent ARM formation. However, the DPM neurons may receive ARM information independently because their fibers are limited within MB lobes and gap-junctional communications between the APL and DPM neurons are specifically required for the formation of ASM, but not ARM. Given that all dopamine reinforcement comes in via the γ KCs, it is possible that the DPM neurons obtain ARM information from γ KCs. Together, these data suggest that two parallel neural pathways, serotoninergic DPM-αβ KCs and octopaminergic APL-α'β' KCs, modulate 3 hr ARM formation in the MB (Wu, 2013).

Additive expression of consolidated memory through Drosophila mushroom body subsets

Associative olfactory memory in Drosophila has two components called labile anesthesia-sensitive memory and consolidated anesthesia-resistant memory (ARM). Mushroom body (MB) is a brain region critical for the olfactory memory and comprised of 2000 neurons that can be classified into αβ, α'β', and γ neurons. It has been previously demonstrated that two parallel pathways mediate ARM consolidation: the serotonergic dorsal paired medial (DPM)-αβ neurons and the octopaminergic anterior paired lateral (APL)-α'β' neurons. This study shows that blocking the output of αβ neurons and that of α'β' neurons each impairs ARM retrieval, and blocking both simultaneously has an additive effect. Knockdown of radish and octβ2R in αβ and α'β' neurons, respectively, impairs ARM. A combinatorial assay of radish mutant background rsh¹ and neurotransmission blockade confirms that ARM retrieved from α'β' neuron output is independent of radish. The MB output neurons MBON-β2β'2a and MBON-β'2mp were identified as the MB output neurons downstream of αβ and α'β' neurons, respectively, whose glutamatergic transmissions also additively contribute to ARM retrieval. Finally, α'β' neurons can be functionally subdivided into α'β'm neurons required for ARM retrieval, and α'β'ap neurons required for ARM consolidation. These data demonstrate that two parallel neural pathways mediating ARM consolidation in Drosophila MB additively contribute to ARM expression during retrieval (Yang, 2016).

The key finding in this study is the identification of two parallel neural pathways that additively express 3-h aversive ARM through Drosophila MB αβ and α'β' neurons. After training, Radish in MB αβ neurons and octopamine signaling in α'β' neurons independently consolidate ARM, which is additively retrieved by αβ-MBON-β2β'2a and α'β'm-MBON-β'2mp circuits for memory expression. Five lines of evidence support this scenario. First, the output from αβ or α'β' neurons is required for ARM retrieval, and the effect of blocking αβ output and that of blocking α'β' output during retrieval are additive. Second, knockdown of radish in αβ neurons, but not in α'β' neurons, impaired ARM, while knockdown of octβ2R in α'β' neurons further impaired the residual ARM in rsh¹ mutant flies. Third, blocking output from α'β' neurons, but not from αβ neurons, during retrieval further impaired the residual ARM in rsh¹ mutant flies. Forth, glutamatergic output from neurons downstream of the αβ or α'β' neurons, i.e., MBON-β2β'2a or MBON-β'2mp neurons, is required for ARM retrieval, and the effects of knockdown of VGlut are additive. Finally, output from α'β'm neurons, but not α'β'ap neurons, is required for ARM retrieval, consistent with the dendritic distribution of MBON-β'2mp neurons (Yang, 2016).

The parallel pathways for 3-h ARM expression were spatially defined by the requirements of neurotransmission from two sets of circuits during retrieval, the αβ-MBON-β2β'2a neurons and the α'β'm-MBON-β'2mp neurons. In addition, blocking neurotransmission from αβ or α'β' neurons during retrieval reduced ARM expression by about 50% whereas simultaneous blockade produced an additive effect that completely abolished ARM expression. Similar additive effects were repeatedly observed in experiments that utilize manipulations in both pathways: an rsh¹ mutant background plus octβ2R RNAi knockdown or plus retrieval blockade in α'β' neurons and knockdown of VGlut in MBON-β2β'2a plus MBON-β'2mp neurons. Thus, total four lines of evidence support the additive expression of 3-h ARM (Yang, 2016).

The parallel pathways for 3-h ARM expression shown in this study differ from the degenerate parallel pathways for the stomatogastric ganglion of the crab or CO₂ avoidance in the fly, as the latter enable mechanisms by which the network output can be switched between states. In the current study, the two parallel neural pathways additively contribute to the expression of 3-h ARM. The nature of the ARM parallel pathways may be similar to that for cold avoidance behavior in the fly, where parallel pathways in the β' and β circuits additively contribute but only the β circuit allows age-dependent alterations for potential benefits against aging (Shih, 2015). Considering the robustness of ARM through the course of senescence, it's unlikely to be age-dependent alterations in ARM system (Yang, 2016).

In studies of Drosophila neurobiology, C305a-GAL4 is a common GAL4 line for α'β' neurons. In this study, by examining three different zoom-in sections of the MB lobes and counting the cells, the following GAL4 lines expressing in α'β' neurons were extensively characterized: VT30604-GAL4 and VT57244-GAL4, which cover most α'β'ap and α'β'm neurons; VT37861-GAL4 and VT50658-GAL4, which cover α'β'ap neurons; and R42D07-GAL4 and R26E01-GAL4, which cover most α'β'm neurons. In contrast, C305a-GAL4 sporadically expresses in about half as many MB neurons as VT30604-GAL4 or VT57244-GAL4 does. Although covering both subsets of α'β' neurons, the expression pattern of C305a-GAL4 in α'β'm neurons is too few and/or weak to lead to a perturbation of synaptic transmission. This is shown by the data that retrieval of 3-h ARM was disrupted by shibire manipulation using all-α'β' neurons driver or α'β'm-specific driver, but neither α'β'ap-specific driver nor C305a-GAL4 for 3-h memory. Note that the GFP signals were acquired from flies carrying two copies of 5XUAS-mCD8::GFP reporter and without any immunostaining-mediated amplification. With the assistance of immunostaining and/or advanced reporter such as increasing copy number of UAS or incorporating a small intron to boost expression, some studies have shown appreciable GFP signal in most α'β' neurons. Given that shibire-mediated neurotransmission blockade and RNAi-mediated knockdown require high enough expression level, the imaging method adopted in this study can faithfully reflect the regions that were effectively manipulated in these behavioral assays. Regarding the pervasive use of C305a-GAL4 for shibire or RNAi manipulation, some functional studies of α'β' neurons might need to be carefully revisited. This study showed, by close examination and cell counting, that VT30604-GAL4, VT37861-GAL4, and R42D07-GAL4 are useful GAL4 lines to study α'β', α'β'ap, and α'β'm neurons, respectively, especially when split-GAL4 lines that span the second and third chromosomes are not genetically feasible (Shih, 2015).

ARM was thought to be diminished in radish mutant flies, in which a truncated RADISH is expressed. It's noteworthy that radish mutants still show a residual 3-h ARM with a PI of roughly 10, which is equal to the 3-h ARM score in wild-type flies fed with an inhibitor of serotonin synthesis to hinder the serotonergic DPM neurotransmission. Interestingly, feeding radish mutant flies with the drug didn't make the 3-h memory score worse, which has already implied that RADISH mediates the consolidation of ARM in the serotonergic DPM-αβ neurons circuit. Indeed, in this study advantage was taken of RNAi-mediated knockdown to identify αβ neurons with RADISH-mediated ARM consolidation. However, only the output from αβs neurons among three subsets of αβ neurons is required for aversive memory retrieval. Whether the αβs neurons are the only aversive ARM substrate of RADISH remains to be identified (Yang, 2016).

APL and DPM neurons are two pairs of modulatory neurons broadly innervating the ipsilateral MB, although the DPM neuron's fiber is lacking in the posterior part of pedunculus and the calyx. Broad, extensive fiber and non-spiking feature allow these two pairs of neurons to have multiple functional roles through different types of neurotransmission. The APL neuron has been shown to receive odor information from the MB neurons and provide GABAergic feedback inhibition as the Drosophila equivalent of a group of the honeybee GABAergic feedback neurons. This feedback inhibition has been proposed to maintain sparse, decorrelated odor coding by suppressing the neuronal activity of MB neurons, which can be somewhat linked to the mutual suppression relation with conditioned odor and the facilitation of reversal learning. Interestingly, Pitman (2011) proposed that the feedback inhibition from APL neurons sustains the labile appetitive ASM based on shibire manipulation. Since shibire manipulation can impact small vesicle release, and APL neurons have been demonstrated to co-release at least GABA and octopamine, it might worth conducting GABA-specific manipulation in APL neurons to confirm the role in appetitive ASM. For aversive olfactory memory, acute RNAi-mediated knockdown of Glutamic acid decarboxylase in APL neurons had no effect on 3-h memory. Instead, the octopamine synthesis enzyme mutant, Tβh^nM18, knockdown of Tβh in APL neurons, the octopamine receptor mutant, PBac{WH}octβ2R^f05679, and knockdown of octβ2R in α'β' neurons all phenocopied the 3-h ARM impairment caused by shibire-mediated neurotransmission blockade in APL neurons. Together with the serotonergic DPM-αβ neurons circuit , a model that is favored that two sets of triple-layered parallel circuits, octopaminergic APL-α'β'-MBON-β'2mp and serotonergic DPM-αβ-MBON-β2β'2a, additively contribute to 3-h aversive ARM (Yang, 2016).

Although the data showed that 3-h ARM consolidation requires recurrent output from α'β'ap neurons but not from α'β'm neurons, RNAi-mediated knockdown of octβ2R in α'β'ap or α'β'm neurons impaired ARM, suggesting that Octβ2R functions for normal ARM expression in the entire population of α'β' neurons. On the other hand, neuronal activity during memory consolidation is naturally more quiescent than that during memory retrieval, and the shibire-mediated neurotransmission blockade requires an exhaustion of already-docked vesicles. Together with the unfavorable performance for experiments blocking the output from α'β'm neurons during consolidation, the possibility cannot be excluded that output from α'β'm neurons is also required for ARM during consolidation. Alternatively, octopamine signaling may also be involved in ARM retrieval (Yang, 2016).

Epigenetic regulator Stuxnet modulates octopamine effect on sleep through a Stuxnet-Polycomb-Octbeta2R cascade

Sleep homeostasis is crucial for sleep regulation. The role of epigenetic regulation in sleep homeostasis is unestablished. Previous studies showed that octopamine is important for sleep homeostasis. However, the regulatory mechanism of octopamine reception in sleep is unknown. This study identified an epigenetic regulatory cascade (Stuxnet-Polycomb-Octβ2R) that modulates the octopamine receptor in Drosophila. stuxnet positively regulates Octβ2R through repression of Polycomb in the ellipsoid body of the adult fly brain and Octβ2R is one of the major receptors mediating octopamine function in sleep homeostasis. In response to octopamine, Octβ2R transcription is inhibited as a result of stuxnet downregulation. This feedback through the Stuxnet-Polycomb-Octβ2R cascade is crucial for sleep homeostasis regulation. This study demonstrates a Stuxnet-Polycomb-Octβ2R-mediated epigenetic regulatory mechanism for octopamine reception, thus providing an example of epigenetic regulation of sleep homeostasis (Zhao, 2021).

Drosophila has been used as a model system to study mechanisms of sleep regulation. The first studies on sleep in Drosophila revealed that they periodically enter a quiescence state that meets a set of criteria for sleep. Drosophila sleep is monitored normally by a Drosophila activity monitoring system (DAMS) and is defined as immobility for 5 min or longer which is a sleep bout. Drosophila sleep mainly happens at night, while a period of siesta is in the mid-day. For example, total sleep time is around 380 min (male) and 250 min (female) during the day time, and 480 min (male) and 490 min (female) during the night time in w1118 (Zhao, 2021).

In Drosophila, central complex structures, especially the ellipsoid body (EB) and fan-shaped body (FSB), are important for sleep homeostasis regulation. Activation of dorsal FSB neurons is sufficient to induce sleep. The dorsal FSB also integrates some sleep inhibiting signals. Both dorsal FSB and EB ring 2 are important in sleep homeostasis. Recently, the helicon cells were found to connect the dorsal FSB and EB Ring 2, indicating that these EB and FSB are connected (Zhao, 2021).

Multiple studies indicate that the epigenetic mechanisms are involved in circadian regulation. However, a direct link between epigenetic regulation and sleep homeostasis is not yet established (Zhao, 2021).

Octopamine (OA) in Drosophila is a counterpart of vertebrate noradrenaline. Previous studies in Drosophila showed that OA is a wake-promoting neurotransmitter and plays an important role in regulating both sleep amount and sleep homeostasis. The mutants of the OA synthesis pathway show an increased total sleep. Activation of OA signaling inhibits sleep homeostasis, while in OA synthesis pathway mutants, an enhanced sleep homeostasis is observed. Study of the neural circuit responsible for the sleep/wake effect of OA showed that octopaminergic ASM neuronsproject to the pars intercerebralis (PI), where OAMB (one of the OA receptors)-expressing insulin-like peptide (ILP)-secreting neurons act as downstream mediators of OA signaling. However, the effects of manipulating ASM neurons or ILP-secreting neurons are much weaker than those observed by manipulating all OA secreting neurons. Moreover, the effect of octopamine is not completely suppressed in the OAMB286 mutant, arguing that another receptor or circuit may participate in this process (Zhao, 2021).

Eight OA receptors are identified to date: OAMB, Octβ1R, Octβ2R, Octβ3R, TAR1, TAR2, TAR3, and Octα2R. Although the expression pattern of OA is identified, the endogenous expression profile of these receptors is lacking. A previous study demonstrated that the mushroom body-expressed OAMB mediates the sleep:wake effect of OA. Recently, Octβ2R was shown to be important for the OA effect on endurance exercise adaptation. How the versatility of OA function is mediated by the diverse array of its receptors needs further study. Moreover, the upstream regulatory mechanisms of OA receptors are still unknown (Zhao, 2021).

A previous study showed that Stuxnet (Stx) is important in mediating Polycomb (Pc) protein degradation in the proteasome (Du, 2016). Stx, which is an ubiquitin like protein, mediates Polycomb (Pc) protein degradation through binding to the proteasome with a UBL domain at its N terminus and to Polycomb through a Pc-binding domain. stx level changes result in a series of homeotic transformation phenotypes. Pc is an epigenetic regulator functioning in Polycomb Group (PcG) Complexes. Although it is reported that PcG component E(Z) is involved in circadian regulation, the role of stx in adult physiological process is unknown (Zhao, 2021).

This study identified the role of the epigenetic regulator stx in sleep regulation. stx positively regulates Octβ2R through regulation of Polycomb in the EB of the adult fly brain. Further study demonstrated that the Stuxnet-Polycomb-Octβ2R cascade plays an important role in sleep regulation. In order to elucidate the role of this Stuxnet-Polycomb-Octβ2R cascade in sleep regulation, the role of various Octβ receptors was systematically identified in sleep regulation. Octβ2R was found to be one of the receptors that mediates OA function in sleep homeostasis. More interestingly, it was found that stx was OA-responsive depending on the Octβ1R. Based on these data, it is proposed that the Stuxnet-Polycomb-Octβ2R cascade provides a feedback mechanism for OA signals to the EB to regulate sleep homeostasis and sleep amount (Zhao, 2021).

This study highlights the importance of epigenetic regulation on sleep. Although epigenetic regulation was intensively studied in adult pathological processes such as cancer, epigenetic factors have been far less studied in other physiological processes such as sleep. This study provides an example of the maintenance role of PcG complex in sleep regulation. Although the core PcG complex component Pc is ubiquitously expressed, its regulator stx is tissue specifically distributed, and this distribution may keep appropriate activity of Pc as well as the PcG complex in a tissue-specific manner. The factors regulating the tissue specificity of stx expression need to be further investigated (Zhao, 2021).

A previous study found that mutation of Octβ2R does not have an obvious sleep phenotype. The current data were compared with the published Octβ2R^f05679 mutant data. Although Octβ2R^f05679 mutant was shown not significantly affected total sleep, this study found that the Octβ2R^f05679 has mild effect on sleep. Other Octβ2R mutants were tested, and it was found that the male flies from these mutations indeed have sleep phenotype (Zhao, 2021).

Published studies have shown that the sleep phenotype of octopamine pathway mutants is different between video-based method and DAM-based method. For example, based on DAM data, the TβH mutant resulted in increased sleep per day, while the same mutant showed decreased sleep based on video data. This study used the video-based method to repeat the TβH mutant phenotype. The results showed that compared with the control flies, the TβH mutant got significantly less sleep. This result is consistent with the previously published data. Through close observation of TβH mutant and control flies, this study found that this mutant has much more frequent grooming behavior than the controls. The TβH mutant and control flies were video recorded for 10 min between ZT3.5 and ZT4.5. The results showed a statistically significant increase of the total number of grooming case. The difference between video-based method and DAM-based method is that these grooming behaviors can be detected in video-based methods, but not in DAM-based methods. Multiple studies have established a positive correlation between octopamine treatment and grooming behavior. Theoretically, TβH allele should result in a decrease in octopamine synthesis. The opposite phenotype may be caused by increased tyramine in TβH mutant or by other feedback regulation. The alleles for Octβ2R receptor used in this study show a similar grooming behavior as the control flies. The previously published octβ2R knockout allele should be a stronger one. The difference of sleep phenotypes between video-based and DAM-based methods may be due to the grooming behavior induced by the massive decrease of octopamine detection. Or other unrelated effects caused by the compensation effect previously reported. One hypothesis is that the significant change of grooming behavior probably masks the sleep behavior. The relationship between grooming and sleep needs to be further clarified. The detection of the sleep phenotype without significant changes in grooming phenotype may be a better strategy to get reliable sleep phenotype. If the increase of grooming in TβH mutant is a side effect caused by the increased tyramine, the identification of the phenotypes of octopamine treatment or collective phenotype of octopamine receptors may be more reliable ways to draw conclusions on the function of octopamine. Furthermore, whether grooming is epistatic to sleep is a problem worthy of further study (Zhao, 2021).

Two aspects of sleep homeostasis need to be further studied. First, this study found that Octβ2R and stx colocalize in a subset of EB neurons. In a previously study, EB R2 neurons were found to be responsible for sleep homeostasis regulation. The relationship of these two groups of EB neurons needs further study. Second, the OA-treated Octβ2R mutant has more sleep recovery than the control. This indicates that OA induces more sleep recovery in the condition of Octβ2R downregulation. It seems that in this condition OA induces certain pathways to counteract its role in sleep homeostasis. One possibility is that Octβ2R negatively regulates Octβ3R which results in increased sleep pressure in the absence of Octβ2R. Further studies are needed to clarify the mechanism (Zhao, 2021).

The results suggest the stx-Pc-Octβ2R regulatory cascade serves as a buffering step for OA function in sleep homeostasis. Two-way regulation of OA on stx leads to reverse changes of stx-the more OA, the less stx and vice versa. Through the function of stx-Pc-Octβ2R regulatory cascade, the Octβ2R transcription is changed accordingly. Variation of Octβ2R transcription could buffer the OA response. As a result, the unfavorable effect of OA causing dramatic decrease of sleep amount and homeostasis could be compensated by its receptor (Zhao, 2021).

The Octopamine receptor Octβ2R regulates ovulation in Drosophila melanogaster

Oviposition is induced upon mating in most insects. Ovulation is a primary step in oviposition, representing an important target to control insect pests and vectors, but limited information is available on the underlying mechanism. This study reports that the beta adrenergic-like octopamine receptor Octβ2R serves as a key signaling molecule for ovulation and recruits Protein kinase A and Ca2+/calmodulin-sensitive kinase II as downstream effectors for this activity. The octβ2r homozygous mutant females are sterile. They displayed normal courtship, copulation, sperm storage and post-mating rejection behavior but are unable to lay eggs. It has been shown previously that octopamine neurons in the abdominal ganglion innervate the oviduct epithelium. Consistently, restored expression of Octβ2R in oviduct epithelial cells is sufficient to reinstate ovulation and full fecundity in the octβ2r mutant females, demonstrating that the oviduct epithelium is a major site of Octβ2R's function in oviposition. It was also found that overexpression of the protein kinase A catalytic subunit or Ca2+/calmodulin-sensitive protein kinase II leads to partial rescue of octβ2r's sterility. This suggests that Octβ2R activates cAMP as well as additional effectors including Ca2+/calmodulin-sensitive protein kinase II for oviposition. All three known β adrenergic-like octopamine receptors stimulate cAMP production in vitro. Octβ1R, when ectopically expressed in the octβ2r's oviduct epithelium, fully reinstated ovulation and fecundity. Ectopically expressed Octβ3R, on the other hand, partly restores ovulation and fecundity while OAMB-K3 and OAMB-AS that increase Ca2+ levels yielded partial rescue of ovulation but not fecundity deficit. These observations suggest that Octβ2R have distinct signaling capacities in vivo and activate multiple signaling pathways to induce egg laying. The findings reported in this study narrow the knowledge gap and offer insight into novel strategies for insect control (Lim, 2014; PubMed).

The octopamine receptor octβ2R is essential for ovulation and fertilization in the fruit fly Drosophila melanogaster

The biogenic monoamine octopamine is essential for ovulation and fertilization in insects. Release of this hormone from neurons in the thoracoabdominal ganglion triggers ovulation and sperm release from the spermathecae. This study shows that the effects of octopamine on ovulation are mediated by at least two different octopamine receptors. In addition to the Oamb receptor that is present in the epithelium of the oviduct, the octβ2R receptor is essential for ovulation and fertilization. Octβ2R is widely expressed in the female reproductive tract. Most prominent is expression in the oviduct muscle and the spermathecae. Animals deficient in expression of the receptor show a severe egg-laying defect. The corresponding females have a much larger ovary that is caused by egg retention in the ovary. Moreover, the very few laid eggs are not fertilized, indicating problems in the process of sperm delivery. It is assumed that octβ2R acts in a similar way as ss2-adrenoreceptors in smooth muscles, were activation of this receptor induces an increase in cAMP levels that lead to relaxation of the muscle. Taken together, these findings show that octopaminergic control of ovulation and fertilization is more complex than anticipated and that various receptors located in different cells act together to enable a well-orchestrated activity of the female reproductive system in response to copulation (Li, 2014).

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 study 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 beta1R (CG6919), DmOct beta2R (CG6989) and DmOct beta3R (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).

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date revised: 25 August 2021

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