Actomyosin contraction in the follicular epithelium provides the major mechanical force for follicle rupture during Drosophila ovulation.

Ovulation is critical for sexual reproduction and consists of the process of liberating fertilizable oocytes from their somatic follicle capsules, also known as follicle rupture. The mechanical force for oocyte expulsion is largely unknown in many species. Our previous work demonstrated that Drosophila ovulation, as in mammals, requires the proteolytic degradation of the posterior follicle wall and follicle rupture to release the mature oocyte from a layer of somatic follicle cells. Here, we identified actomyosin contraction in somatic follicle cells as the major mechanical force for follicle rupture. Filamentous actin (F-actin) and nonmuscle myosin II (NMII) are highly enriched in the cortex of follicle cells upon stimulation with octopamine (OA), a monoamine critical for Drosophila ovulation. Pharmacological disruption of F-actin polymerization prevented follicle rupture without interfering with the follicle wall breakdown. In addition, we demonstrated that OA induces Rho1 guanosine triphosphate (GTP)ase activation in the follicle cell cortex, which activates Ras homolog (Rho) kinase to promote actomyosin contraction and follicle rupture. All these results led us to conclude that OA signaling induces actomyosin cortex enrichment and contractility, which generates the mechanical force for follicle rupture during Drosophila ovulation. Due to the conserved nature of actomyosin contraction, this work could shed light on the mechanical force required for follicle rupture in other species including humans.

A neurotransmitter produced by gut bacteria modulates host sensory behaviour.

Animals coexist in commensal, pathogenic or mutualistic relationships with complex communities of diverse organisms, including microorganisms1. Some bacteria produce bioactive neurotransmitters that have previously been proposed to modulate nervous system activity and behaviours of their hosts2,3. However, the mechanistic basis of this microbiota-brain signalling and its physiological relevance are largely unknown. Here we show that in Caenorhabditis elegans, the neuromodulator tyramine produced by commensal Providencia bacteria, which colonize the gut, bypasses the requirement for host tyramine biosynthesis and manipulates a host sensory decision. Bacterially produced tyramine is probably converted to octopamine by the host tyramine ß-hydroxylase enzyme. Octopamine, in turn, targets the OCTR-1 octopamine receptor on ASH nociceptive neurons to modulate an aversive olfactory response. We identify the genes that are required for tyramine biosynthesis in Providencia, and show that these genes are necessary for the modulation of host behaviour. We further find that C. elegans colonized by Providencia preferentially select these bacteria in food choice assays, and that this selection bias requires bacterially produced tyramine and host octopamine signalling. Our results demonstrate that a neurotransmitter produced by gut bacteria mimics the functions of the cognate host molecule to override host control of a sensory decision, and thereby promotes fitness of both the host and the microorganism.

Octopamine metabolically reprograms astrocytes to confer neuroprotection against α-synuclein.

Octopamine is a well-established invertebrate neurotransmitter involved in fight or flight responses. In mammals, its function was replaced by epinephrine. Nevertheless, it is present at trace amounts and can modulate the release of monoamine neurotransmitters by a yet unidentified mechanism. Here, through a multidisciplinary approach utilizing in vitro and in vivo models of α-synucleinopathy, we uncovered an unprecedented role for octopamine in driving the conversion from toxic to neuroprotective astrocytes in the cerebral cortex by fostering aerobic glycolysis. Physiological levels of neuron-derived octopamine act on astrocytes via a trace amine-associated receptor 1-Orai1-Ca2+-calcineurin-mediated signaling pathway to stimulate lactate secretion. Lactate uptake in neurons via the monocarboxylase transporter 2-calcineurin-dependent pathway increases ATP and prevents neurodegeneration. Pathological increases of octopamine caused by α-synuclein halt lactate production in astrocytes and short-circuits the metabolic communication to neurons. Our work provides a unique function of octopamine as a modulator of astrocyte metabolism and subsequent neuroprotection with implications to α-synucleinopathies.

Octopamine in the mushroom body circuitry for learning and memory.

Octopamine, the functional analog of noradrenaline, modulates many different behaviors and physiological processes in invertebrates. In the central nervous system, a few octopaminergic neurons project throughout the brain and innervate almost all neuropils. The center of memory formation in insects, the mushroom bodies, receive octopaminergic innervations in all insects investigated so far. Different octopamine receptors, either increasing or decreasing cAMP or calcium levels in the cell, are localized in Kenyon cells, further supporting the release of octopamine in the mushroom bodies. In addition, different mushroom body (MB) output neurons, projection neurons, and dopaminergic PAM cells are targets of octopaminergic neurons, enabling the modulation of learning circuits at different neural sites. For some years, the theory persisted that octopamine mediates rewarding stimuli, whereas dopamine (DA) represents aversive stimuli. This simple picture has been challenged by the finding that DA is required for both appetitive and aversive learning. Furthermore, octopamine is also involved in aversive learning and a rather complex interaction between these biogenic amines seems to modulate learning and memory. This review summarizes the role of octopamine in MB function, focusing on the anatomical principles and the role of the biogenic amine in learning and memory.

Sensitivity to expression levels underlies differential dominance of a putative null allele of the Drosophila tßh gene in behavioral phenotypes.

The biogenic amine octopamine (OA) and its precursor tyramine (TA) are involved in controlling a plethora of different physiological and behavioral processes. The tyramine-ß-hydroxylase (tßh) gene encodes the enzyme catalyzing the last synthesis step from TA to OA. Here, we report differential dominance (from recessive to overdominant) of the putative null tßhnM18 allele in 2 behavioral measures in Buridan's paradigm (walking speed and stripe deviation) and in proboscis extension (sugar sensitivity) in the fruit fly Drosophila melanogaster. The behavioral analysis of transgenic tßh expression experiments in mutant and wild-type flies as well as of OA and TA receptor mutants revealed a complex interaction of both aminergic systems. Our analysis suggests that the different neuronal networks responsible for the 3 phenotypes show differential sensitivity to tßh gene expression levels. The evidence suggests that this sensitivity is brought about by a TA/OA opponent system modulating the involved neuronal circuits. This conclusion has important implications for standard transgenic techniques commonly used in functional genetics.

Past experience shapes sexually dimorphic neuronal wiring through monoaminergic signalling.

Differences between female and male brains exist across the animal kingdom and extend from molecular to anatomical features. Here we show that sexually dimorphic anatomy, gene expression and function in the nervous system can be modulated by past experiences. In the nematode Caenorhabditis elegans, sexual differentiation entails the sex-specific pruning of synaptic connections between neurons that are shared by both sexes, giving rise to sexually dimorphic circuits in adult animals1. We discovered that starvation during juvenile stages is memorized in males to suppress the emergence of sexually dimorphic synaptic connectivity. These circuit changes result in increased chemosensory responsiveness in adult males following juvenile starvation. We find that an octopamine-mediated starvation signal dampens the production of serotonin (5-HT) to convey the memory of starvation. Serotonin production is monitored by a 5-HT1A serotonin receptor homologue that acts cell-autonomously to promote the pruning of sexually dimorphic synaptic connectivity under well-fed conditions. Our studies demonstrate how life history shapes neurotransmitter production, synaptic connectivity and behavioural output in a sexually dimorphic circuit.

A gut microbial factor modulates locomotor behaviour in Drosophila.

While research into the biology of animal behaviour has primarily focused on the central nervous system, cues from peripheral tissues and the environment have been implicated in brain development and function1. There is emerging evidence that bidirectional communication between the gut and the brain affects behaviours including anxiety, cognition, nociception and social interaction1-9. Coordinated locomotor behaviour is critical for the survival and propagation of animals, and is regulated by internal and external sensory inputs10,11. However, little is known about how the gut microbiome influences host locomotion, or the molecular and cellular mechanisms involved. Here we report that germ-free status or antibiotic treatment results in hyperactive locomotor behaviour in the fruit fly Drosophila melanogaster. Increased walking speed and daily activity in the absence of a gut microbiome are rescued by mono-colonization with specific bacteria, including the fly commensal Lactobacillus brevis. The bacterial enzyme xylose isomerase from L. brevis recapitulates the locomotor effects of microbial colonization by modulating sugar metabolism in flies. Notably, thermogenetic activation of octopaminergic neurons or exogenous administration of octopamine, the invertebrate counterpart of noradrenaline, abrogates the effects of xylose isomerase on Drosophila locomotion. These findings reveal a previously unappreciated role for the gut microbiome in modulating locomotion, and identify octopaminergic neurons as mediators of peripheral microbial cues that regulate motor behaviour in animals.

A platform utilizing Drosophila ovulation for nonhormonal contraceptive screening.

A significant unmet need for new contraceptive options for both women and men remains due to side-effect profiles, medical concerns, and the inconvenience of many currently available contraceptive products. Unfortunately, the development of novel nonsteroidal female contraceptive medicine has been stalled in the last couple of decades due to the lack of effective screening platforms. Drosophila utilizes conserved signaling pathways for follicle rupture, a final step in ovulation that is essential for female reproduction. Therefore, we explored the potential to use Drosophila as a model to screen compounds that could inhibit follicle rupture and be nonsteroidal contraceptive candidates. Using our ex vivo follicle rupture assay, we screened 1,172 Food and Drug Administration (FDA)-approved drugs and identified six drugs that could inhibit Drosophila follicle rupture in a dose-dependent manner. In addition, we characterized the molecular actions of these drugs in the inhibition of adrenergic signaling and follicle rupture. Furthermore, we validated that three of the four drugs consistently inhibited mouse follicle rupture in vitro and that two of them did not affect progesterone production. Finally, we showed that chlorpromazine, one of the candidate drugs, can significantly inhibit mouse follicle rupture in vivo. Our work suggests that Drosophila ovulation is a valuable platform for identifying lead compounds for nonsteroidal contraceptive development and highlights the potential of these FDA-approved drugs as novel nonsteroidal contraceptive agents.

Short-term exposure to high pCO2 leads to decreased branchial cytochrome C oxidase activity in the presence of octopamine in a decapod.

In a recent mechanistic study, octopamine was shown to promote proton transport over the branchial epithelium in green crabs, Carcinus maenas. Here, we follow up on this finding by investigating the involvement of octopamine in an environmental and physiological context that challenges acid-base homeostasis, the response to short-term high pCO2 exposure (400 Pa) in a brackish water environment. We show that hyperregulating green crabs experienced a respiratory acidosis as early as 6 h of exposure to hypercapnia, with a rise in hemolymph pCO2 accompanied by a simultaneous drop of hemolymph pH. The slightly delayed increase in hemolymph HCO3- observed after 24 h helped to restore hemolymph pH to initial values by 48 h. Circulating levels of the biogenic amine octopamine were significantly higher in short-term high pCO2 exposed crabs compared to control crabs after 48 h. Whole animal metabolic rates, intracellular levels of octopamine and cAMP, as well as branchial mitochondrial enzyme activities for complex I + III and citrate synthase were unchanged in posterior gill #7 after 48 h of hypercapnia. However, application of octopamine in gill respirometry experiments suppressed branchial metabolic rate in posterior gills of short-term high pCO2 exposed animals. Furthermore, branchial enzyme activity of cytochrome C oxidase decreased in high pCO2 exposed crabs after 48 h. Our results indicate that hyperregulating green crabs are capable of quickly counteracting a hypercapnia-induced respiratory acidosis. The role of octopamine in the acclimation of green crabs to short-term hypercapnia seems to entail the alteration of branchial metabolic pathways, possibly targeting mitochondrial cytochrome C in the gill. Our findings help advancing our current limited understanding of endocrine components in hypercapnia acclimation. SUMMARY STATEMENT: Acid-base compensation upon short-term high pCO2 exposure in hyperregulating green crabs started after 6 h and was accomplished by 48 h with the involvement of the biogenic amine octopamine, accumulation of hemolymph HCO3-, and regulation of mitochondrial complex IV (cytochrome C oxidase).

Neural Regulatory Pathways of Feeding and Fat in Caenorhabditis elegans.

The compact nervous system of Caenorhabditis elegans and its genetic tractability are features that make this organism highly suitable for investigating energy balance in an animal system. Here, we focus on molecular components and organizational principles emerging from the investigation of pathways that largely originate in the nervous system and regulate feeding behavior but also peripheral fat regulation through neuroendocrine signaling. We provide an overview of studies aimed at understanding how C. elegans integrate internal and external cues in feeding behavior. We highlight some of the similarities and differences in energy balance between C. elegans and mammals. We also provide our perspective on unresolved issues, both conceptual and technical, that we believe have hampered critical evaluation of findings relevant to fat regulation in C. elegans.

Tyramine beta hydroxylase-mediated octopamine synthesis pathway in Litopenaeus vannamei under thermal, salinity, and Vibrio alginolyticus infection stress.

Stress responses impact the immune systems, growth, and reproduction of aquatic organisms. Neuroendocrine regulation involving biogenic amines, including octopamine (OA), plays a pivotal role in maintaining physiological balance during stress. This study focuses on the synthesis pathway of OA, particularly the role of tyramine beta hydroxylase (TBH), in Litopenaeus vannamei under stress. TBH catalyzes the conversion of tyramine to OA, a process critical for physiological responses. The present study demonstrated LvTBH at the protein level under different stress conditions during acute (0.5, 1, 2 h) and chronic stress (24, 72, 168 h) periods. LvTBH increased in thoracic ganglia within 2 h under hyperthermal stress, accompanied by elevated OA levels. Conversely, LvTBH decreased in the brain and circumesophageal connective tissues during acute and chronic hypothermal stress. Additionally, LvTBH increased in the brain and circumesophageal connective tissues under acute infection stress, coinciding with elevated OA levels. These findings collectively contribute to a more intricate understanding of the neuroendocrine dynamics within L. vannamei under stress, underscoring the role of TBH in orchestrating responses crucial for adaptation.

Octopamine neuron dependent aggression requires dVGLUT from dual-transmitting neurons.

Neuromodulators such as monoamines are often expressed in neurons that also release at least one fast-acting neurotransmitter. The release of a combination of transmitters provides both "classical" and "modulatory" signals that could produce diverse and/or complementary effects in associated circuits. Here, we establish that the majority of Drosophila octopamine (OA) neurons are also glutamatergic and identify the individual contributions of each neurotransmitter on sex-specific behaviors. Males without OA display low levels of aggression and high levels of inter-male courtship. Males deficient for dVGLUT solely in OA-glutamate neurons (OGNs) also exhibit a reduction in aggression, but without a concurrent increase in inter-male courtship. Within OGNs, a portion of VMAT and dVGLUT puncta differ in localization suggesting spatial differences in OA signaling. Our findings establish a previously undetermined role for dVGLUT in OA neurons and suggests that glutamate uncouples aggression from OA-dependent courtship-related behavior. These results indicate that dual neurotransmission can increase the efficacy of individual neurotransmitters while maintaining unique functions within a multi-functional social behavior neuronal network.

Molecular characterization and functional roles for Vibrio alginolyticus resistance of an octopamine/tyramine receptor of the white shrimp, Litopenaeus vannamei.

Octopamine and Tyramine are biogenic amines that have been demonstrated to play an important immunological role in white shrimp, Litopenaeus vannamei. G protein-coupled receptors, known as seven-transmembrane domain receptors, are a variety of neurotransmitter receptors which are sensitive to biogenic amines for initiating the cell signaling pathway. In present study, we cloned and characterized an octopamine/tyramine receptor (LvOA/TA-R) from the hemocytes of L. vannamei, with a 1194 b.p. open reading frame that encodes 398 amino acids. Several bioinformatics analyses indicated that LvOA/TA-R had seven conserved hydrophobic transmembrane domains. The phylogenetic analysis and multiple sequence alignment indicated that LvOA/TA-R was orthologous to the OA/TA receptor of tiger shrimp, P. monodon. LvOA/TA-R was expressed in hemocytes and nervous tissue including circumoesphageal connective tissue and the thoracic and abdominal ganglia. Significant increases in LvOA/TA-R occurred in hemocytes of L. vannamei under Vibrio alginolyticus infection within 30-60 min of infection. Here, we demonstrated that LvOA/TA-R expression is upregulated in response to Vibrio alginolyticus infection and appears to be functionally responsible for the observed immune response. These results suggest that LvOA/TA-R mediates regulation of immunity, which promotes the resistance of L. vannamei to V. alginolyticus.

Neuromodulators signal through astrocytes to alter neural circuit activity and behaviour.

Astrocytes associate with synapses throughout the brain and express receptors for neurotransmitters that can increase intracellular calcium (Ca2+). Astrocytic Ca2+ signalling has been proposed to modulate neural circuit activity, but the pathways that regulate these events are poorly defined and in vivo evidence linking changes in astrocyte Ca2+ levels to alterations in neurotransmission or behaviour is limited. Here we show that Drosophila astrocytes exhibit activity-regulated Ca2+ signalling in vivo. Tyramine and octopamine released from neurons expressing tyrosine decarboxylase 2 (Tdc2) signal directly to astrocytes to stimulate Ca2+ increases through the octopamine/tyramine receptor (Oct-TyrR) and the transient receptor potential (TRP) channel Water witch (Wtrw), and astrocytes in turn modulate downstream dopaminergic neurons. Application of tyramine or octopamine to live preparations silenced dopaminergic neurons and this inhibition required astrocytic Oct-TyrR and Wtrw. Increasing astrocyte Ca2+ signalling was sufficient to silence dopaminergic neuron activity, which was mediated by astrocyte endocytic function and adenosine receptors. Selective disruption of Oct-TyrR or Wtrw expression in astrocytes blocked astrocytic Ca2+ signalling and profoundly altered olfactory-driven chemotaxis and touch-induced startle responses. Our work identifies Oct-TyrR and Wtrw as key components of the astrocytic Ca2+ signalling machinery, provides direct evidence that octopamine- and tyramine-based neuromodulation can be mediated by astrocytes, and demonstrates that astrocytes are essential for multiple sensory-driven behaviours in Drosophila.

Feeding state sculpts a circuit for sensory valence in Caenorhabditis elegans.

Hunger affects the behavioral choices of all animals, and many chemosensory stimuli can be either attractive or repulsive depending on an animal's hunger state. Although hunger-induced behavioral changes are well documented, the molecular and cellular mechanisms by which hunger modulates neural circuit function to generate changes in chemosensory valence are poorly understood. Here, we use the CO2 response of the free-living nematode Caenorhabditis elegans to elucidate how hunger alters valence. We show that CO2 response valence shifts from aversion to attraction during starvation, a change that is mediated by two pairs of interneurons in the CO2 circuit, AIY and RIG. The transition from aversion to attraction is regulated by biogenic amine signaling. Dopamine promotes CO2 repulsion in well-fed animals, whereas octopamine promotes CO2 attraction in starved animals. Biogenic amines also regulate the temporal dynamics of the shift from aversion to attraction such that animals lacking octopamine show a delayed shift to attraction. Biogenic amine signaling regulates CO2 response valence by modulating the CO2-evoked activity of AIY and RIG. Our results illuminate a new role for biogenic amine signaling in regulating chemosensory valence as a function of hunger state.

Tyramine action on motoneuron excitability and adaptable tyramine/octopamine ratios adjust Drosophila locomotion to nutritional state.

Adrenergic signaling profoundly modulates animal behavior. For example, the invertebrate counterpart of norepinephrine, octopamine, and its biological precursor and functional antagonist, tyramine, adjust motor behavior to different nutritional states. In Drosophila larvae, food deprivation increases locomotor speed via octopamine-mediated structural plasticity of neuromuscular synapses, whereas tyramine reduces locomotor speed, but the underlying cellular and molecular mechanisms remain unknown. We show that tyramine is released into the CNS to reduce motoneuron intrinsic excitability and responses to excitatory cholinergic input, both by tyraminehonoka receptor activation and by downstream decrease of L-type calcium current. This central effect of tyramine on motoneurons is required for the adaptive reduction of locomotor activity after feeding. Similarly, peripheral octopamine action on motoneurons has been reported to be required for increasing locomotion upon starvation. We further show that the level of tyramine-ß-hydroxylase (TBH), the enzyme that converts tyramine into octopamine in aminergic neurons, is increased by food deprivation, thus selecting between antagonistic amine actions on motoneurons. Therefore, octopamine and tyramine provide global but distinctly different mechanisms to regulate motoneuron excitability and behavioral plasticity, and their antagonistic actions are balanced within a dynamic range by nutritional effects on TBH.

Molecular and Pharmacological Characterization of ß-Adrenergic-like Octopamine Receptors in the Endoparasitoid Cotesia chilonis (Hymenoptera: Braconidae).

Octopamine (OA) is structurally and functionally similar to adrenaline/noradrenaline in vertebrates, and OA modulates diverse physiological and behavioral processes in invertebrates. OA exerts its actions by binding to specific octopamine receptors (OARs). Functional and pharmacological characterization of OARs have been investigated in several insects. However, the literature on OARs is scarce for parasitoids. Here we cloned three ß-adrenergic-like OARs (CcOctßRs) from Cotesia chilonis. CcOctßRs share high similarity with their own orthologous receptors. The transcript levels of CcOctßRs were varied in different tissues. When heterologously expressed in CHO-K1 cells, CcOctßRs induced cAMP production, and were dose-dependently activated by OA, TA and putative octopaminergic agonists. Their activities were inhibited by potential antagonists and were most efficiently blocked by epinastine. Our study offers important information about the molecular and pharmacological properties of ß-adrenergic-like OARs from C. chilonis that will provide the basis to reveal the contribution of individual receptors to the physiological processes and behaviors in parasitoids.

PaOctß2R: Identification and Functional Characterization of an Octopamine Receptor Activating Adenylyl Cyclase Activity in the American Cockroach Periplaneta americana.

Biogenic amines constitute an important group of neuroactive substances that control and modulate various neural circuits. These small organic compounds engage members of the guanine nucleotide-binding protein coupled receptor (GPCR) superfamily to evoke specific cellular responses. In addition to dopamine- and 5-hydroxytryptamine (serotonin) receptors, arthropods express receptors that are activated exclusively by tyramine and octopamine. These phenolamines functionally substitute the noradrenergic system of vertebrates Octopamine receptors that are the focus of this study are classified as either α- or ß-adrenergic-like. Knowledge on these receptors is scarce for the American cockroach (Periplaneta americana). So far, only an α-adrenergic-like octopamine receptor that primarily causes Ca2+ release from intracellular stores has been studied from the cockroach (PaOctα1R). Here we succeeded in cloning a gene from cockroach brain tissue that encodes a ß-adrenergic-like receptor and leads to cAMP production upon activation. Notably, the receptor is 100-fold more selective for octopamine than for tyramine. A series of synthetic antagonists selectively block receptor activity with epinastine being the most potent. Bioinformatics allowed us to identify a total of 19 receptor sequences that build the framework of the biogenic amine receptor clade in the American cockroach. Phylogenetic analyses using these sequences and receptor sequences from model organisms showed that the newly cloned gene is an ß2-adrenergic-like octopamine receptor. The functional characterization of PaOctß2R and the bioinformatics data uncovered that the monoaminergic receptor family in the hemimetabolic P. americana is similarly complex as in holometabolic model insects like Drosophila melanogaster and the honeybee, Apis mellifera. Thus, investigating these receptors in detail may contribute to a better understanding of monoaminergic signaling in insect behavior and physiology.

Octopaminergic neurons have multiple targets in Drosophila larval mushroom body calyx and can modulate behavioral odor discrimination.

Discrimination of sensory signals is essential for an organism to form and retrieve memories of relevance in a given behavioral context. Sensory representations are modified dynamically by changes in behavioral state, facilitating context-dependent selection of behavior, through signals carried by noradrenergic input in mammals, or octopamine (OA) in insects. To understand the circuit mechanisms of this signaling, we characterized the function of two OA neurons, sVUM1 neurons, that originate in the subesophageal zone (SEZ) and target the input region of the memory center, the mushroom body (MB) calyx, in larval Drosophila We found that sVUM1 neurons target multiple neurons, including olfactory projection neurons (PNs), the inhibitory neuron APL, and a pair of extrinsic output neurons, but relatively few mushroom body intrinsic neurons, Kenyon cells. PN terminals carried the OA receptor Oamb, a Drosophila α1-adrenergic receptor ortholog. Using an odor discrimination learning paradigm, we showed that optogenetic activation of OA neurons compromised discrimination of similar odors but not learning ability. Our results suggest that sVUM1 neurons modify odor representations via multiple extrinsic inputs at the sensory input area to the MB olfactory learning circuit.

Concerted Actions of Octopamine and Dopamine Receptors Drive Olfactory Learning.

Aminergic signaling modulates associative learning and memory. Substantial advance has been made in Drosophila on the dopamine receptors and circuits mediating olfactory learning; however, our knowledge of other aminergic modulation lags behind. To address this knowledge gap, we investigated the role of octopamine in olfactory conditioning. Here, we report that octopamine activity through the ß-adrenergic-like receptor Octß1R drives aversive and appetitive learning: Octß1R in the mushroom body αß neurons processes aversive learning, whereas Octß1R in the projection neurons mediates appetitive learning. Our genetic interaction and imaging studies pinpoint cAMP signaling as a key downstream effector for Octß1R function. The rutabaga-adenylyl cyclase synthesizes cAMP in a Ca2+/calmodulin-dependent manner, serving as a coincidence detector for associative learning and likely representing a downstream target for Octß1R. Supporting this notion, the double heterozygous rutabaga/+;octß1r/+ flies perform poorly in both aversive and appetitive conditioning, while individual heterozygous rutabaga/+ and octß1r/+ flies behave like the wild-type control. Consistently, the mushroom body and projection neurons in the octß1r brain exhibit blunted responses to octopamine when cAMP levels are monitored through the cAMP sensor. We previously demonstrated the pivotal functions of the D1 receptor dDA1 in aversive and appetitive learning, and the α1 adrenergic-like receptor OAMB in appetitive learning. As expected, octß1r genetically interacts with dumb (dDA1 mutant) in aversive and appetitive learning, but it interacts with oamb only in appetitive learning. This study uncovers the indispensable contributions of dopamine and octopamine signaling to aversive and appetitive learning. All experiments were performed on mixed sex unless otherwise noted.SIGNIFICANCE STATEMENT Animals make flexible behavioral choices that are constantly shaped by experience. This plasticity is vital for animals to appropriately respond to the cues predicting benefit or harm. In Drosophila, dopamine is known to mediate both reward-based and punishment-based learning while octopamine function is important only for reward. Here, we demonstrate that the octopamine-Octß1R-cAMP pathway processes both aversive and appetitive learning in distinct neural sites of the olfactory circuit. Furthermore, we show that the octopamine-Octß1R and dopamine-dDA1 signals together drive both aversive and appetitive learning, whereas the octopamine-Octß1R and octopamine-OAMB pathways jointly facilitate appetitive, but not aversive, learning. This study identifies the cognate actions of octopamine and dopamine signaling as a key neural mechanism for associative learning.