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.

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.

Bite force, body size, and octopamine mediate mating interactions in the house cricket (Acheta domesticus).

Mating interactions are rife with conflict because the evolutionary interests of males and females seldom coincide. Intersexual conflict affects sexual selection, yet the proximate factors underlying male coercive ability and female resistance are poorly understood. Male combat outcomes are often influenced by bite force, with superior biters being more likely to achieve victory over poorer biters in a range of species, including crickets. If good performers also achieve mating success through sexual coercion, then bite force might play a role in intersexual conflict as well. We tested the capacity of bite force to influence mating interactions in house crickets both directly by measuring bite forces of males and females and by altering male bite capacity through neuropharmacological manipulation. In addition, the invertebrate neurotransmitter octopamine both mediates aggression and underlies motivation to bite in male house crickets. By blocking octopamine receptors through the application of an antagonist, epinastine, we tested the effects of reduced bite force on male mating success. Our results show that male bite capacity, in combination with body size, influences both the likelihood and the outcomes of mating interactions, whereas treatment of males with epinastine eliminates motivation to mate. Our results suggest a functional role for bite force in affecting both sexual conflict and sexual selection and expand our knowledge of the influence of biogenic amines on reproductive behaviour.

Epigenetic regulator Stuxnet modulates octopamine effect on sleep through a Stuxnet-Polycomb-Octß2R 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. In this study, we identify an epigenetic regulatory cascade (Stuxnet-Polycomb-Octß2R) that modulates the octopamine receptor in Drosophila. We demonstrate that stuxnet positively regulates Octß2R through repression of Polycomb in the ellipsoid body of the adult fly brain and that Octß2R is one of the major receptors mediating octopamine function in sleep homeostasis. In response to octopamine, Octß2R transcription is inhibited as a result of stuxnet downregulation. This feedback through the Stuxnet-Polycomb-Octß2R cascade is crucial for sleep homeostasis regulation. This study demonstrates a Stuxnet-Polycomb-Octß2R-mediated epigenetic regulatory mechanism for octopamine reception, thus providing an example of epigenetic regulation of sleep homeostasis.

Visuo-Motor Feedback Modulates Neural Activities in the Medulla of the Honeybee, Apis mellifera.

Behavioral and internal-state modulation of sensory processing has been described in several organisms. In insects, visual neurons in the optic lobe are modulated by locomotion, but the degree to which visual-motor feedback modulates these neurons remains unclear. Moreover, it also remains unknown whether self-generated and externally generated visual motion are processed differently. Here, we implemented a virtual reality system that allowed fine-scale control over visual stimulation in relation to animal motion, in combination with multichannel recording of neural activity in the medulla of a female honeybee (Apis mellifera). We found that this activity was modulated by locomotion, although, in most cases, only when the bee had behavioral control over the visual stimulus (i.e., in a closed-loop system). Moreover, closed-loop control modulated a third of the recorded neurons, and the application of octopamine (OA) evoked similar changes in neural responses that were observed in a closed loop. Additionally, in a subset of modulated neurons, fixation on a visual stimulus was preceded by an increase in firing rate. To further explore the relationship between neuromodulation and adaptive control of the visual environment of the bee, we modified motor gain sensitivity while locally injecting an OA receptor antagonist into the medulla. Whereas female honeybees were tuned to a motor gain of -2 to 2 (between the heading of the bee and its visual feedback), local disruption of the OA pathway in the medulla abolished this tuning, resulting in similar low levels of response across levels of motor gain. Our results show that behavioral control modulates neural activity in the medulla and ultimately impacts behavior.SIGNIFICANCE STATEMENT When moving, an animal generates the motion of the visual scene over its retina. We asked whether self-generated and externally generated optic flow are processed differently in the insect medulla. Our results show that closed-loop control of the visual stimulus modulates neural activity as early as the medulla and ultimately impacts behavior. Moreover, blocking octopaminergic modulation further disrupted object-tracking responses. Our results suggest that the medulla is an important site for context-dependent processing of visual information and that placing the animal in a closed-loop environment may be essential to understanding its visual cognition and processing.

Octopamine affects courtship call structure in male Acheta domesticus crickets.

Secondary sexual displays vary considerably in both type and structure both within and across animal species. Although such variation is of keen interest to evolutionary biologists, the functional factors driving variation in male displays are poorly understood. In crickets, acoustic calls are produced by muscular contractions via stridulation of file and scraper wing components. We tested the effect of varying octopamine, an important biogenic amine neurohormone in invertebrates, on call production in male Acheta domesticus house crickets by blocking the octopamine receptors that influence skeletal muscle function with epinastine, a synthetic octopamine antagonist. We then measured male courtship calls and analyzed the call structure to quantify the differences in call structure based on the changes in carrier frequency, and whether chirps or ticks are a more prevalently expressed frequency in treated vs untreated males. Males treated with epinastine exhibited clear differences in call structure compared to untreated controls, such that epinastine-treated males were more likely to produce simpler calls and to exhibit their carrier frequencies as ticks rather than chirps. Thus, we were able to directly modify male courtship calling performance during mating interactions by altering the neuropharmacological milieu, demonstrating the potential role of biogenic amines in contributing to the diversity of call types in nature.

Octopamine modulates insect mating and Oviposition.

The neuro-mechanisms that regulate insect reproduction are not fully understood. Biogenic amines, including octopamine, are neuromodulators that have been shown to modulate insect reproduction in various ways, e.g., promote or inhibit insect mating or oviposition. In this study, we examined the role of octopamine in regulating the reproduction behaviors of a devastating underground insect pest, the dark black chafer (Holotrichia parallela). We first measured the abundance of octopamine in different neural tissues of the adult chafer pre- and post-mating, demonstrating that octopamine decreased in the abdominal ganglia of females but increased in males post-mating. We then fed the adult H. parallela with a concentration gradient of octopamine to test the effects on insect reproductive behaviors. Compared with its antagonist mianserin, octopamine at the concentration of 2 µg/mL resulted in the highest increase in males' preference for sex pheromone and females' oviposition, whereas the mianserin-treatment increased the survival rate and prolonged the lifespan of H. parallela. In addition, we did not observe significant differences in egg hatchability between octopamine and mianserin-treated H. parallela. Our results demonstrated that octopamine promotes H. parallela mating and oviposition with a clear low dosage effect, illustrated how neural substrates modulate insect behaviors, and provided insights for applying octopamine in pest management.

Reciprocal modulation of 5-HT and octopamine regulates pumping via feedforward and feedback circuits in C. elegans.

Feeding is vital for animal survival and is tightly regulated by the endocrine and nervous systems. To study the mechanisms of humoral regulation of feeding behavior, we investigated serotonin (5-HT) and octopamine (OA) signaling in Caenorhabditis elegans, which uses pharyngeal pumping to ingest bacteria into the gut. We reveal that a cross-modulation mechanism between 5-HT and OA, which convey feeding and fasting signals, respectively, mainly functions in regulating the pumping and secretion of both neuromodulators via ADF/RIC/SIA feedforward neurocircuit (consisting of ADF, RIC, and SIA neurons) and ADF/RIC/AWB/ADF feedback neurocircuit (consisting of ADF, RIC, AWB, and ADF neurons) under conditions of food supply and food deprivation, respectively. Food supply stimulates food-sensing ADFs to release more 5-HT, which augments pumping via inhibiting OA secretion by RIC interneurons and, thus, alleviates pumping suppression by OA-activated SIA interneurons/motoneurons. In contrast, nutrient deprivation stimulates RICs to secrete OA, which suppresses pumping via activating SIAs and maintains basal pumping and 5-HT production activity through excitation of ADFs relayed by AWB sensory neurons. Notably, the feedforward and feedback circuits employ distinct modalities of neurosignal integration, namely, disinhibition and disexcitation, respectively.

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.

Enhancement of synaptic responses in ascending interneurones following acquisition of social dominance in crayfish.

When crayfish have attained dominant status after agonistic bouts, their avoidance reaction to mechanical stimulation of the tailfan changes from a dart to a turn response. Ascending interneurones originating in the terminal ganglion receive sensory inputs from the tailfan and they affect spike activity of both uropod and abdominal postural motor neurones, which coordinates the uropod and abdominal postural movements. Despite the varying output effects of ascending interneurones, the synaptic responses of all interneurones to sensory stimulation were enhanced when they acquired a dominant state. The number of spikes increased as did a sustained membrane depolarizations. Regardless of social status, the output effects on the uropod motor neurones of all interneurones except VE-1 remained unchanged. VE-1 mainly inhibited the uropod opener motor neurones in naive animals, but tended to excite them in dominant animals. Synaptic enhancement of the sensory response of ascending interneurones was also observed in naive animals treated with bath-applied serotonin. However, subordinate animals or naive animals treated with octopamine had no noticeable effect on the synaptic response of their ascending interneurones to sensory stimulation. Thus, enhancement of the synaptic response is a specific neural event that occurs when crayfish attain social dominance and it is mediated by serotonin.

Opposing effects of dopamine on agonistic behaviour in crayfish.

The effects of dopamine on the agonistic behaviour of crayfish were analysed. When dopamine concentrations of 1 µmol l-1 were injected into large crayfish, individuals were beaten by smaller opponents, despite their physical advantage. Injection of 10 µmol l-1 dopamine into small animals increased their rate of winning against larger opponents. Injection of a D1 receptor antagonist prohibited the onset of a 'loser' effect in subordinate animals, suggesting that the inhibitory effect of dopamine on larger animals is mediated by D1 receptors. Similarly, injection of a D2 receptor antagonist prohibited the onset of a 'winner' effect in dominant animals, suggesting that the facilitating effect of dopamine on small animals is mediated by D2 receptors. Since the inhibitory effect of 1 µmol l-1 dopamine was similar to that seen with 1 µmol l-1 octopamine and the facilitating effect of 10 µmol l-1 dopamine was similar to that of 1 µmol l-1 serotonin, functional interactions among dopamine, octopamine and serotonin were analyzed by co-injection of amines with their receptor antagonists in various combinations. The inhibitory effect of 1 µmol l-1 dopamine disappeared when administered with D1 receptor antagonist, but remained when combined with octopamine receptor antagonist. Octopamine effects disappeared when administered with either D1 receptor antagonist or octopamine receptor antagonist, suggesting that the dopamine system is downstream of octopamine. The facilitating effect of 10 µmol l-1 dopamine disappeared when combined with serotonin 5HT1 receptor antagonist or D2 receptor antagonist. Serotonin effects also disappeared when combined with D2 receptor antagonist, suggesting that dopamine and serotonin activate each other through parallel pathways.

Variation in mobility and exercise adaptations between Drosophila species.

Locomotion and mobility have been studied extensively in Drosophila melanogaster but less is known about the locomotor capacity of other Drosophila species, while the response to chronic exercise in other species has yet to be examined. We have shown that adult male D. melanogaster adapt to exercise training with improved running endurance, climbing speed, and flight ability compared to unexercised flies. Here, we examine baseline mobility of D. sechellia, D. simulans, and D. virilis, and their response to chronic exercise training. We found significant interspecific differences in mobility and in the response to exercise. Although there is a significant sex difference in exercise adaptations in D. melanogaster, intraspecific analysis reveals few sex differences in other Drosophila species. As octopamine has been shown to be important for exercise adaptations in D. melanogaster, we also asked if any observed differences could be attributed to baseline octopamine levels. We find that octopamine and tyramine levels have the same rank order as baseline climbing speed and endurance in males, but do not predict the response to chronic exercise in males or females. Future research should focus on determining the mechanisms responsible for the inter- and intraspecific differences in mobility and the response to exercise.

p-Synephrine, ephedrine, p-octopamine and m-synephrine: Comparative mechanistic, physiological and pharmacological properties.

Confusion and misunderstanding exist regarding the lack of cardiovascular and other adverse health effects of p-synephrine and p-octopamine relative to ephedrine and m-synephrine (phenylephrine) which are known for their effects on the cardiovascular system. These four molecules have some structural similarities. However, the structural and stereochemical differences of p-synephrine and p-octopamine as related to ephedrine and m-synephrine result in markedly different adrenergic receptor binding characteristics as well as other mechanistic differences which are reviewed. p-Synephrine and p-octopamine exhibit little binding to α-1, α-2, ß-1 and ß-2 adrenergic receptors, nor are they known to exhibit indirect actions leading to an increase in available levels of endogenous norepinephrine and epinephrine at commonly used doses. The relative absence of these mechanistic actions provides an explanation for their lack of production of cardiovascular effects at commonly used oral doses as compared to ephedrine and m-synephrine. As a consequence, the effects of ephedrine and m-synephrine cannot be directly extrapolated to p-synephrine and p-octopamine which exhibit significantly different pharmacokinetic, and physiological/pharmacological properties. These conclusions are supported by human, animal and in vitro studies that are discussed.

Differential modulation of courtship behavior and subsequent aggression by octopamine, dopamine and serotonin in male crickets.

Aggression is a behavioral strategy for securing limited resources and its expression is strongly influenced by their presence and value. In particular, males are generally thought to guard females after mating to ward off other males, but the underlying control mechanisms are unknown. Here, we investigated the role of amines on male courtship behavior and its subsequent effect on male-male aggression in crickets (Gryllus bimaculatus). Contrary to the guarding hypothesis, female presence alone had no immediate effect on male-male aggression. Furthermore, confirming studies on other species, prior female contact, but not necessarily courtship or copulation, promoted subsequent male-male aggression in subordinate, but not socially naive crickets. This promoting effect of female contact is transient and slowly wanes after her removal. Selective aminergic receptor antagonists revealed that the promoting effect of prior female contact on male-male aggression is mediated by octopamine (OA), as well as by serotonin (5HT) acting most likely via 5HT1 and/or 5HT7 like receptors. This contrasts the role of 5HT2-like receptors in maintaining reduced aggressiveness after social defeat. Furthermore, while dopamine (DA) is necessary for the recovery of aggression in subordinates after defeat, it appears to play no part in female induced aggression. Male courtship, on the other hand, is selectively promoted by DA and 5HT, again most likely via 5HT1 and/or 5HT7 like receptors, but not by OA. We conclude that OA, DA and 5HT each differentially modulate different aspects of courtship and aggressive behavior in a context specific fashion.

Transient enhancement of immune resistance functions in Litopenaeus vannamei through a low-dose octopamine injection.

Octopamine (OA) is known to play an important role in regulating invertebrate immune responses. In this study, we determined the effects of OA on immunity and physiological regulation in the white shrimp Litopenaeus vannamei. The total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase (PO) activity, respiratory bursts (RBs), superoxide dismutase (SOD) activity, and lysozyme, glucose, and lactate levels in plasma, and phagocytic activity and clearance efficiency in response to the pathogen, Vibrio alginolyticus, were measured when shrimp (11.1-13.0 g) were individually injected with saline or OA at 100 and 1000 pmol shrimp-1. Results showed significant increases in THC, semigranular cells (SGCs), and PO activity per 50 µL of haemolymph at 0.5-4 h; granular cells at 0.5-2 h; respiratory bursts (RBs) at 0.5-1 h; phagocytic activity at 2-4 h; and clearance efficiency at 2-8 h, but PO activity per granulocyte at 0.5-2 h significantly decreased after the OA injection. All of the immune parameters had returned to control values by 8 h after receiving OA except granular cells at 4 h, RBs at 2 h, clearance efficiency at 16 h, and PO activity per granulocyte at 4 h. However, no significant differences were observed in hyaline cells, RBs per haemocyte, lysozyme and SOD activities, glucose, or lactate during the experimental period. An injection of OA also significantly decreased the mortality of shrimp challenged with V. alginolyticus. In another experiment, the immune-related genes of transglutaminase-I, lipopolysaccharide- and ß-1,3-glucan-binding protein, prophenoloxidase-II, and peroxidase of shrimp that received 1000 pmol OA shrimp-1 for 1 h were significantly higher than those of shrimp that received the saline control. These results suggest that OA administration at ≤1000 pmol shrimp-1 mediates transient upregulation of immunity, which in turn promotes the resistance of L. vannamei to V. alginolyticus.

Pharmacological Properties of the Type 1 Tyramine Receptor in the Diamondback Moth, Plutella xylostella.

Tyramine receptors (TARs) can be activated by tyramine (TA) or octopamine (OA) and have been shown to be related to physiological regulation (e.g., gustatory responsiveness, social organization, and learning behavior) in a range of insect species. A tyramine receptor gene in Plutella xylostella, Pxtar1, was cloned and stably expressed in the HEK-293 cell line. Pharmacological properties and expression profile of Pxtar1 were also analyzed. Tyramine could activate the PxTAR1 receptor, increasing the intracellular Ca2+ concentration ((Ca2+)i) at an EC50 of 13.1 nM and reducing forskolin (10 µM)-stimulated intracellular cAMP concentration ((cAMP)i) at an IC50 of 446 nM. DPMF (a metabolite of amitraz) and L(-)-carvone (an essential oil) were found to act as PxTAR1 receptor agonists. Conversely, yohimbine and mianserin had significant antagonistic effects on PxTAR1. In both larvae and adults, Pxtar1 had the highest expression in the head capsule and expression of Pxtar1 was higher in male than in female reproductive organs. This study reveals the temporal and spatial differences and pharmacological properties of Pxtar1 in P. xylostella and provides a strategy for screening insecticidal compounds that target PxTAR1.

Octopamine enhances oxidative stress resistance through the fasting-responsive transcription factor DAF-16/FOXO in C. elegans.

Dietary restriction regimens lead to enhanced stress resistance and extended life span in many species through the regulation of fasting and/or diet-responsive mechanisms. The fasting stimulus is perceived by sensory neurons and causes behavioral and metabolic adaptations. Octopamine (OA), one of the Caenorhabditis elegans neurotransmitters, is involved in behavioral adaptations, and its levels are increased under fasting conditions. However, it remains largely unknown how OA contributes to the fasting responses. In this study, we found that OA administration enhanced organismal resistance to oxidative stress. This enhanced resistance was suppressed by a mutation of the OA receptors, SER-3 and SER-6. Moreover, we found that OA administration promoted the nuclear translocation of DAF-16, the key transcription factor in fasting responses, and that the OA-induced enhancement of stress resistance required DAF-16. Altogether, our results suggest that OA signaling, which is triggered by the absence of food, shifts the organismal state to a more protective one to prepare for environmental stresses.

Tyraminergic modulation of agonistic outcomes in crayfish.

Octopamine, a biogenic amine, modulates various behaviors, ranging from locomotion and aggression to learning and memory in invertebrates. Several studies recently demonstrated that tyramine, the biological precursor of octopamine, also affects behaviors independent of octopamine. Here we investigated the involvement of tyramine in agonistic interaction of the male crayfish Procambarus clarkii. When male crayfish fight, larger animals (3-7% difference in body length) are more likely to win. By contrast, direct injection of tyramine or octopamine counteracted the physical advantage of larger animals. Tyramine or octopamine-injected naive large animals were mostly beaten by untreated smaller naive animals. This pharmacological effect was similar to the loser effect in which subordinate larger animals are frequently beaten by smaller animals. Furthermore, loser effects were partly eliminated by either injection of epinastine, an octopamine blocker, or yohimbine, a tyramine blocker, and significantly diminished by injection of a mixture of both blockers. We also observed that tyramine levels in the subesophageal ganglion were remarkably increased in subordinate crayfish after losing a fight. These results suggest that tyramine modulates aggressive levels of crayfish and contributes to the loser effect in parallel with octopamine.

Visual discrimination transfer and modulation by biogenic amines in honeybees.

For more than a century, visual learning and memory have been studied in the honeybee Apis mellifera using operant appetitive conditioning. Although honeybees show impressive visual learning capacities in this well-established protocol, operant training of free-flying animals cannot be combined with invasive protocols for studying the neurobiological basis of visual learning. In view of this, different attempts have been made to develop new classical conditioning protocols for studying visual learning in harnessed honeybees, though learning performance remains considerably poorer than that for free-flying animals. Here, we investigated the ability of honeybees to use visual information acquired during classical conditioning in a new operant context. We performed differential visual conditioning of the proboscis extension reflex (PER) followed by visual orientation tests in a Y-maze. Classical conditioning and Y-maze retention tests were performed using the same pair of perceptually isoluminant chromatic stimuli, to avoid the influence of phototaxis during free-flying orientation. Visual discrimination transfer was clearly observed, with pre-trained honeybees significantly orienting their flights towards the former positive conditioned stimulus (CS+), thus showing that visual memories acquired by honeybees are resistant to context changes between conditioning and the retention test. We combined this visual discrimination approach with selective pharmacological injections to evaluate the effect of dopamine and octopamine in appetitive visual learning. Both octopaminergic and dopaminergic antagonists impaired visual discrimination performance, suggesting that both these biogenic amines modulate appetitive visual learning in honeybees. Our study brings new insight into cognitive and neurobiological mechanisms underlying visual learning in honeybees.

Conditional modulation of spike-timing-dependent plasticity for olfactory learning.

Mushroom bodies are a well-known site for associative learning in insects. Yet the precise mechanisms that underlie plasticity there and ensure their specificity remain elusive. In locusts, the synapses between the intrinsic mushroom body neurons and their postsynaptic targets obey a Hebbian spike-timing-dependent plasticity (STDP) rule. Although this property homeostatically regulates the timing of mushroom body output, its potential role in associative learning is unknown. Here we show in vivo that pre-post pairing causing STDP can, when followed by the local delivery of a reinforcement-mediating neuromodulator, specify the synapses that will undergo an associative change. At these synapses, and there only, the change is a transformation of the STDP rule itself. These results illustrate the multiple actions of STDP, including a role in associative learning, despite potential temporal dissociation between the pairings that specify synaptic modification and the delivery of reinforcement-mediating neuromodulator signals.