InteractiveFly: GeneBrief

Tyramine β hydroxylase: Biological Overview | References


Gene name - Tyramine β hydroxylase

Synonyms -

Cytological map position - 7D2-7D2

Function - enzyme

Keywords - response to ethanol, Gonads, octopamine biosynthesis, memory; larval locomotory behavior; regulation of forward locomotion, aggression

Symbol - Tbh

FlyBase ID: FBgn0010329.html

Genetic map position - X:7,889,730..7,920,567 [+]

Classification - Copper type II ascorbate-dependent monooxygenase and dopamine beta-monooxygenase N-terminal) domain

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Hasan, M. N., Hosen, M. J., Thakur, P. K., Abir, R. A., Zubaer, A., Renkai, G., Yoshida, M., Ohta, H., Lee, J. M., Kusakabe, T. and Hirashima, A. (2016). In vitro screening for inhibitor of cloned Drosophila melanogaster tyramine-β-hydroxylase and docking studies. Int J Biol Macromol 93: 889-895. PubMed ID: 27355756
Summary:
Biogenic amines are common biologically active substances extended within the whole animal kingdom where they play vital roles as signal transducer as well as regulator of cell functions. One of these biogenic amines called octopamine (OA) is synthesized from tyramine (TA) by the catalysis of tyramine-β-hydroxylase (TβH) originated in the insect nervous system. Both TA and OA act as neurotransmitters, neurohormones and neuromodulators in the arthropod nervous system. In this study, the inhibitory activity of 1-arylimidazole-2(3H)-thiones (AITs) was tested on cloned Drosophila tyramine-β-hydroxylase (DmTβH) expressed in Bombyx mori strain. Radiolabelled 3H-TA was used to analyze the activity of AITs exhibited inhibitory effects on DmTβH, whose ID50 values range from 0.02 to 2511nM where DmTβH was inhibited in a dose-dependent manner at pH 7.6 and 25 ° C during a 30min of incubation. To understand the catalytic role of the TβH, a three dimensional structure of the TβH from Drosophila melanogaster was constructed by homology modeling using the Phyre2 web server with 100% confidence. The modeled three-dimensional structure of TβH was used to perform the docking study with AITs. This may give more insights to precise design of inhibitors for TβH to control insect's population.
Iliadi, K. G., Iliadi, N. and Boulianne, G. L. (2017). Drosophila mutants lacking octopamine exhibit impairment in aversive olfactory associative learning. Eur J Neurosci [Epub ahead of print]. PubMed ID: 28715094
Summary:
Octopamine is a biogenic amine in invertebrates that is considered a functional homolog of vertebrate norepinephrine. Octopamine regulates many physiological processes such as metabolism, reproduction and different types of behaviour including learning and memory. Previous studies in insects led to the notion that acquisition of an olfactory memory depends on the octopaminergic system during appetitive (reward-based) learning, but not in the case of aversive (punishment-based) learning. This study provides several lines of evidence demonstrating that aversive associative olfactory learning in Drosophila is also dependent on octopamine signaling. Specifically, Drosophila Tβh (Tyramine-β-hydroxylase) mutants, which lack octopamine and are female sterile, were examined to determine whether octopamine plays a role in aversive learning. Tβh mutant flies are shown to exhibit a significant reduction in learning compared to control lines that is independent of either genetic background or the methods used to induce aversive olfactory memory. To unambiguously demonstrate that octopamine synthesis plays a role in aversive olfactory learning, rescue experiments were performed using the Gal4/UAS system. Expression of UAS-Tβh in octopamine/tyraminergic neurons using Tdc2-Gal4 in Tβh null mutant flies fully rescued both the aversive learning defects and female sterility observed in Tβh mutants.
Damrau, C., Toshima, N., Tanimura, T., Brembs, B. and Colomb, J. (2017). Octopamine and tyramine contribute separately to the counter-regulatory response to sugar deficit in Drosophila. Front Syst Neurosci 11: 100. PubMed ID: 29379421
Summary:
All animals constantly negotiate external with internal demands before and during action selection. Energy homeostasis is a major internal factor biasing action selection. For instance, in addition to physiologically regulating carbohydrate mobilization, starvation-induced sugar shortage also biases action selection toward food-seeking and food consumption behaviors (the counter-regulatory response). Biogenic amines are often involved when such widespread behavioral biases need to be orchestrated. In mammals, norepinephrine (noradrenalin) is involved in the counterregulatory response to starvation-induced drops in glucose levels. The invertebrate homolog of noradrenalin, octopamine (OA) and its precursor tyramine (TA) are neuromodulators operating in many different neuronal and physiological processes. Tyrosine-β-hydroxylase (tβh) mutants are unable to convert TA into OA. It was hypothesized that tβh mutant flies may be aberrant in some or all of the counter-regulatory responses to starvation and that techniques restoring gene function or amine signaling may elucidate potential mechanisms and sites of action. Corroborating this hypothesis, starved mutants show a reduced sugar response and their hemolymph sugar concentration is elevated compared to control flies. When starved, they survive longer. Temporally controlled rescue experiments revealed an action of the OA/TA-system during the sugar response, while spatially controlled rescue experiments suggest actions also outside of the nervous system. Additionally, the analysis of two OA- and four TA-receptor mutants suggests an involvement of both receptor types in the animals' physiological and neuronal response to starvation.
BIOLOGICAL OVERVIEW

In mammals and humans, noradrenaline is a key modulator of aggression. Octopamine, a closely related biogenic amine, has been proposed to have a similar function in arthropods. However, the effect of octopamine on aggressive behavior is little understood. An automated video analysis of aggression in male Drosophila has been developed, rendering aggression accessible to high-throughput studies. The software detects the lunge, a conspicuous behavioral act unique to aggression. In lunging, the aggressor rears up on his hind legs and snaps down on his opponent. By using the software to eliminate confounding effects, this study shows that aggression is almost abolished in mutant males lacking octopamine. The mutant used was TβhnM18, which lack tyramine β-hydroxylase (TβH), an enzyme converting tyramine (TA) to octopamine (OA). This suppression is independent of whether tyramine, the precursor of octopamine, is increased or also depleted. Restoring octopamine synthesis in the brain either throughout life or in adulthood leads to a partial rescue of aggression. Finally, neuronal silencing of octopaminergic and tyraminergic neurons almost completely abolishes lunges. It is concluded that octopamine modulates Drosophila aggression. Genetically depleting the animal of octopamine downregulates lunge frequency without a sizable effect on the lunge motor program. This study provides access to the neuronal circuitry mediating this modulation (Hoyer, 2008).

Vertebrates require noradrenaline to display aggression. For example, dopamine β-hydroxylase knockout mice lacking noradrenaline hardly show any aggressive behavior (Marino, 2005). The effect of noradrenaline is suggested to be biphasic: Slight increases in noradrenaline level lead to enhanced aggressive behavior, whereas strong elevations suppress aggression. Less is known about the role of octopamine (OA) in arthropod aggression, but its effects seem to be equally complex. In crustaceans, OA injection leads to a submissive-looking body posture. In crickets, injection of the OA agonist chlordimeform causes normally submissive losers of fights to re-engage in fighting faster than sham-injected animals. Likewise in honeybees, injection of two OA agonists, XAMI and DCDM, biases the likelihood of aggressive display toward non-nestmates over nestmates. In Drosophila, agonistic encounters of males and females are composed of a variety of both offensive and defensive components, some of which are displayed more often in one sex than in the other. For example, 'lunging,' i.e., rearing on the hind legs and snapping down on the opponent, is characteristic of males, whereas 'low posture fencing,' i.e., pushing each other with the legs, is displayed by both genders. Up to now, two studies investigated the role of OA in Drosophila aggression. Both used a mutant for tyramine β-hydroxylase (TβH), an enzyme converting tyramine (TA) to OA. Mutant TβhnM18 flies lacked OA but showed about 10-fold-increased TA levels in the brain (Monastirioti, 1996). Taking various aggressive behavioral components into account, Baier (2002) observed in fights between white-eyed TβhnM18 and wild-type males a decrease of aggressive behavioral patterns in the mutant. In contrast, focusing on the males' behavioral choice between aggression and courtship, Certel (2007) did not report a general decrease in aggression for TβhnM18 males when fighting against each other. However, if males approached other males by vibrating their wing(s), which occurred in about three encounters per 30 min recording period, TβhnM18 males less often showed a transition to aggressive behavior than did wild-type males (Hoyer, 2008).

This study reports on an automated recording of Drosophila male aggression that allows a high throughput under standardized conditions. The software detects one of the key features of aggression: the lunge. With this tool it was demonstrated that (1) small differences in body size influence the outcome of a fight in favor of the larger male, (2) walking activity correlates positively with lunge frequency, and (3) flies mutant for the white gene, a member of the ABC transporter gene family, are profoundly impaired in aggression not only because of the deteriorated optics of their eyes but also due to the missing gene function in the central nervous system. Excluding the influences of these factors that had confounded a previous study (Baier, 2002), this study shows that males without OA display hardly any lunge behavior, even though execution of the lunge motor program is largely indistinguishable from that of wild-type males. Presumably, an elaborate pattern of OA, and possibly TA, levels in time and space is required to enable flies to express wild-type aggressive behavior (Hoyer, 2008).

Quantifying the rich repertoire of Drosophila aggressive behavior by manually evaluating and interpreting video recordings is a time-consuming and demanding task. Therefore an automated evaluation tool was developed that detects a single, distinct component of Drosophila male aggression, the lunge, in video clips of Drosophila behavior. The lunge is a striking feature of male aggression that does not occur in other behavioral contexts. Within a lunge, three phases can be distinguished. During the first phase, the attacking fly rises on his hind legs, lifting his long body axis by 49.2 ± 1.2°. He then snaps down on his opponent (phase 2), with his head reaching a velocity of 254 ± 11.8 mm/s and his body reaching forces of about twice his body mass. Finally, the attacking male tries to grab his opponent with his forelegs and, if successful, pulls him toward his own body (phase 3; not always present (Hoyer, 2008).

To have the software identify lunges in image sequences, it was essential to confine flies to a horizontal arena surrounded by high glass walls covered with Fluon, rendering the walls too slippery for flies to hold on. In this way, overall aggression was high because flies could not avoid further encounters. All encounters were recorded (Hoyer, 2008).

The software program developed for this study records the number of lunges for each fly in a certain time interval. In addition, it provides information such as the distance the fly walked, his size, and the time he spent on the food patch and in the periphery. Because the lunge has been reported to be the most frequent behavior by which an opponent is displaced from the food patch, the number of lunges of a male may serve, at least to some extent, as an indicator of his overall aggressiveness (Hoyer, 2008).

To evaluate the reliability of the software, the same clips were analyzed twice with respect to the number of lunges: once by the software and once 'by hand.' The software is designed to minimize false-positive assignments (counting frame sequences wrongly as lunges). This leads to a slightly larger number of false negatives (missing lunges). The software underestimates the occurrence of lunges by about 11%. This value is independent of the lunge frequency. Importantly, it is also largely independent of genotype. Only if a genotype results in a high percentage of nonfighting males does the overall error rate differ from that of wild-type because for nonfighting males, the number of lunges can only be overestimated (Hoyer, 2008).

Overestimating lunge frequency for nonfighting males can hide subtle differences between genotypes. Therefore, a 'lunge view' software program was added that enables the investigator to focus only on those frame sequences that contain lunges according to the 'lunge count' software. The investigator can then decide whether the selected frame sequences indeed represent lunges, thereby eliminating false positives (Hoyer, 2008).

To determine baseline aggressive behavior of wild-type flies in this paradigm, CantonS (CS) males were tested. Independent of the time of day, a pair of five-day-old CS males performed 3.85 ± 2.82 lunges/min, demonstrating the high variability already observed in other paradigms of Drosophila male aggression. In the present study, aggression was recorded from the 15th to the 30th min, constituting a period when flies already had settled into the arena and displayed constant aggression at a level indistinguishable from that of the two subsequent 15 min time bins (Hoyer, 2008).

The total number of lunges performed by a pair of males correlated positively with their overall walked distance, i.e., the more the two flies walked the more lunges they performed. This correlation could be demonstrated for numerous genotypes. The pairs of flies were regarded as one unit (total lunges and total distance walked) because the interactions were strongly dependent upon both flies. Not just the dominant fly approached the subordinate one; the subordinate fly often returned to the food patch, thereby eliciting new attacks. A decision was made to normalize lunge frequency to walking activity for two reasons: (1) variance was strongly reduced by this step. A pair of five-day-old CS males performed 16.4 ± 6.6 lunges/m. (2) In mutant studies differences in lunge frequency between genotypes might be a side effect of differences in walking activity rather than a result of alterations in aggressiveness. However, because walking activity and aggression might be regulated by separate mechanisms, the lunge count software allows for evaluation of the two separately, if necessary (Hoyer, 2008).

The two males did not lunge equally often within the recording period. In 156 of 172 pairs that performed at least 10 lunges, one male performed more than 70% of all lunges. As in many other species, in Drosophila the size difference between two males strongly influences which male wins more aggressive encounters. The effect is most obvious when the weight difference between the opponents is pronounced (~50%). These data show that a size difference of just 8% (measured as the projection area from above) results in the bigger fly being likely to lunge more often than the smaller fly. Because the 8% difference in body size cannot be detected by the human eye, fights were always set up between males of the same genotype in order to avoid a confounding influence of size when investigating the effect of a specific genotype (Hoyer, 2008).

Many transgenic fly lines are generated and kept in a white mutant background. Therefore, the role was examined of the white (w) gene in aggressive behavior. Males mutant for the null allele w1118 were strongly impaired in aggression, lunging at a rate of only 3% of wild-type male levels. Providing w1118 males with a mini-white+ transgene had differing effects but never resulted in a full rescue of wild-type aggression (Hoyer, 2008).

Mutant w1118 flies lacking the characteristic red pigmentation of the eyes are visually impaired. Indeed, an intact visual system is required for normal aggressive behavior, as blind norpAP24 hemizygote and motion-blind homozygous ninaE17 males performed significantly fewer lunges per meter than wild-type Berlin (WT-B) males. Consequently, it was asked whether to show aggression males needed the white gene function in vision for proper pattern contrast in the eye. For a tissue-specific knockdown, the eye-specific GMR-GAL4 line was used to drive a UAS-RNAi-white transgene. These males showed only a light coloring of the adult eye, and aggression was almost completely abolished. In an inverse experiment, the eye-color phenotype was rescued in males carrying a GMR-white construct in a w1118 mutant background. Interestingly, with flies fighting at 28%-65% of wild-type level, the aggression was only partially restored independent of the number of constructs and their location. This suggests that an intact visual system is required for proper aggressive behavior. Because the flies' eye colors were dark red but still clearly distinguishable from wild-type CS males, this experiment did not rule out that the lower-than-WT level of aggression reflected an incomplete restoration of contrast transfer in these eyes (Hoyer, 2008).

In contrast, white gene function might be required in tissues of the fly other than the pigment-producing cells in the eye. The latter idea is supported by findings of Campbell (2001), who detected white messenger RNA in so1 flies by using RT-PCR. Mutant so1 flies have neither eyes nor ocelli and should therefore lack pigment-producing cells. Also, in a place-learning paradigm in complete darkness (heat box), w1118 null mutant flies are impaired. To test whether the white mutation affects neurons outside the eye, various GAL4 drivers (Ddc-GAL4; TH-GAL4, Tdc2-GAL4, MB247-GAL4; NP6510-GAL4, NP6561-GAL4) expressing GAL4 in groups of neurons in the central brain were combined with the UAS-RNAi-white transgene. Indeed, diminishing white expression in these cells reduced the frequency of lunges to varying degrees ranging from 5%-48% of wild-type level. These results suggest that white exerts its effect not only in pigment-producing cells but also in other parts of the brain, some of which are involved in the control of aggression (Hoyer, 2008).

Scoring various components of Drosophila aggressive behavior, Baier (2002) report severely reduced aggression in TβhnM18 males. TβhnM18 mutant flies lack tyramine β-hydroxylase (TβH), an enzyme converting tyramine (TA) to octopamine (OA). These flies have no detectable levels of OA, whereas TA levels are elevated by about 10-fold (Monastirioti, 1996). These authors, however, had used TβhnM18 males carrying the additional w1118 null mutant allele and tested them with red-eyed control males. As shown above, the w1118 null mutation by itself leads to profoundly reduced aggression. Furthermore, even after backcrossing the TβhnM18 flies to w+, mutant males were still about 8% smaller than wild-type males. Hence, the body-size difference might have contributed to the decreased aggression as well. To test whether reduced aggression was indeed due to the TβhnM18 mutation and independent of body size, it was measured in pairs of mutant males and in the automated recording setup counting only lunges. Aggression was still almost completely abolished (Hoyer, 2008).

In contrast to these results, Certel (2007) did not report a general decrease in aggression compared to wild-type males when TβhnM18 males fought against each other. To exclude genetic background as the cause for this discrepancy (their TβhnM18 mutant stock had been independently crossed into w+ background, Certel, 2007) their stock was tested in the current paradigm. These males displayed profoundly fewer lunges per meter compared to wild-type males. However, with a remaining level of 17% of wild-type, males of their TβhnM18 mutant stock were more aggressive than males of the current TβhnM18 mutant stock, which displayed hardly any aggressive behaviour (Hoyer, 2008).

On the basis of published effects of OA, two hypotheses were tested that might explain the strong decrease in aggression observed for TβhnM18 males. (1) During jumping, distance and force production of TβhnM18 flies is only ~50%-60% of wild-type level (Zumstein, 2004). Consequently, TβhnM18 males might be incapable of executing lunges. However, a quantitative high-speed analysis measuring 12 parameters of lunges did reveal only a single small difference between lunges of CS and TβhnM18 males: While rising up on their hind legs, TβhnM18 males did not elevate their body as much as wild-type males (-26%; p = 0.005). In other words, only the frequency, but not the execution, of lunges seemed to be affected. (2) Injection of the OA agonist chlordimeform into crickets causes normally submissive losers to re-engage in fights faster (Stevenson, 2005). Therefore, appropriate levels of OA might be required to motivate former losers to fight again. If TβhnM18 males establish a hierarchy within the first 15 min and the loser thereafter avoids to re-engage in further fights, lunges might become a rare event. To test this hypothesis, the first 15 min immediately after pairing the flies were analyzed. Right from the beginning, TβhnM18 males performed hardly any lunges, indicating a general loss of aggressiveness independent of former experiences (Hoyer, 2008).

Whether restoring OA in TβhnM18 males would increase the frequency of lunges was investigated. This would strengthen the assumption that it is indeed the lack of OA that elicits the low-aggression phenotype. TβhnM18 females are sterile, and fecundity can be restored by feeding octopamine (Monastirioti, 1996; Monastirioti, 2003). Moreover, feeding OA successfully rescues a memory deficit of TβhnM18 flies (Schwaerzel, 2003). In that study, OA should have crossed the insect blood-brain barrier because it was supposed to have its effect in the mushroom body, a structure of the central brain. Five mg/ml OA was provided in normal fly food either throughout the whole life span or only during adult life. Neither treatment restored aggression in TβhnM18 males compared to wild-type males. The same feeding protocol, however, reverted female sterility independent of the onset of OA supplement, indicating that OA was ingested and still active in the fly (Hoyer, 2008).

TβhnM18 males carrying a wild-type Tβh cDNA downstream of the hsp70 promoter (hsp-Tβh) were used to show that the Tβh locus is responsible for the behavioral changes measured here. The heat-shock protocol applied had already been used successfully to rescue the above-mentioned memory deficit of TβhnM18 flies (Schwaerzel, 2003). Heat-shock-induced expression of Tβh in adult TβhnM18 males restored aggression to a small but significant extent compared to both males of the same genotype without heat shock and to heat-shocked TβhnM18 males lacking the hsp-Tβh construct. 47% of all mutant TβhnM18 pairs that temporarily expressed TβH in all cells showed at least one lunge, whereas only 14% and 9% of all pairs of the same genotype without heat shock and of TβhnM18 males lacking the hsp-Tβh construct, respectively, showed at least one lunge. This result substantiates the role of octopamine in modulating Drosophila male aggression. Because this partial rescue was hidden in the noise of the software, clips were evaluated manually (Hoyer, 2008).

To rescue fecundity in females, a slightly stronger heat-shock protocol was applied. It resulted in a percentage of Tβh;; hsp-Tβh egg-laying females that were indistinguishable from wild-type (Hoyer, 2008).

The rather poor performance of TβhnM18 males that temporarily expressed TβH in all cells might be due to the short time window in which TβH was expressed. In the light of immunohistochemical data indicating that there are neurons expressing TA, but not OA (Nagaya, 2002), misexpression of TβH, alternatively, might change tyraminergic into octopaminergic neurons, which might have deleterious effects on aggression (Hoyer, 2008).

Because OA-supplemented food did not rescue aggression in TβhnM18 males, whether the increased TA, rather than the lack of OA in TβhnM18 males, might have caused the aggression phenotype was examined. To address this issue, mutants of the tyrosine decarboxylase 2 (Tdc2) gene (Tdc2RO54) were used. Tyrosine decarboxylase catalyzes the first step in octopamine biosynthesis. Tyrosine decarboxylase 2 converts tyrosine to TA in neurons. HPLC measurements reveal no detectable levels of TA and OA in Tdc2RO54 mutant brains (Cole, 2005). Males homozygous for a mutation in the nearby cinnabar gene (cn1) were used as a control because the Tdc2RO54 mutant also carried it. Tdc2RO54cn1 males were strongly reduced in aggression compared to Tdc2RO54cn1 heterozygote males and to cn1 males. With the lunge count software, it was determined that their lunge frequency was at about 5% of control levels. This result strongly suggests that in Tdc2RO54 and TβhnM18 males it is indeed the missing OA that causes the aggression phenotype. TA could only still be held responsible if too little TA was as deleterious for aggression as too much (Hoyer, 2008).

Providing mutant Tdc2RO54 males with TA/OA-supplemented food during adulthood again did not restore aggression. The same feeding protocol, however, rescued Tdc2RO54 female sterility. The applied protocol has been demonstrated to restore brain TA and OA levels of Tdc2RO54 mutant flies to wild-type levels (Hardie, 2007). Interestingly, Hardie reported that feeding only TA could not restore OA levels, 'as if ectopically supplied amines were not transported into the appropriate neurons where the metabolic conversion could take place.' To ensure restoration of OA and TA levels within neurons, UAS-Tdc was expressed in all tyraminergic and octopaminergic neurons by using Tdc2-GAL4. There are two genes encoding for a TDC in flies: Tdc1 is expressed nonneuronally and Tdc2 in neurons only (Cole, 2005). Surprisingly, not Tdc2 expression, but Tdc1 expression in Tdc2-neurons yielded a small but significant rescue of aggression compared with Tdc2RO54 males carrying either only the Tdc2-GAL4 transgene or the UAS-Tdc1 construct. Tdc2RO54, Tdc2-GAL4;UAS-Tdc1 males lunged at a rate of 3% compared to the heterozygote controls, whereas Tdc2RO54 males very rarely displayed a lunge (Hoyer, 2008).

In general, the aggressive behavior displayed was highly variable. Two separately collected datasets were pooled in this study; in one of the two experiments Tdc2RO54, Tdc2-GAL4;UAS-Tdc1 males were only significantly different to one control. In accordance with previous reports (Cole, 2005; Hardie, 2007) and with the current findings on aggression, Tdc1 expression seemed to be more potent in rescuing female sterility than Tdc2 expression, with the latter restoring female fecundity only partially. Strikingly, expressing UAS-Tdc2 yielded higher OA and TA levels than did expressing UAS-Tdc1; in fact, TA levels were even higher than in wild-type flies (Cole, 2005). Possibly, Drosophila male aggression is sensitive to deviations from wild-type OA/TA concentrations, resulting in suppressed aggression (Hoyer, 2008).

Feeding wild-type flies OA (5mg/ml) or TA (0.3 mg/ml) did not affect aggression. Also, overexpression of Tβh with the hs-TβH transgene had no effect on lunge frequency. This finding argues that in the TβhnM18 mutant it is not the excess of TA that is deleterious. Also in the rescue experiments above, the small or missing effects could not be attributed to too high levels of OA or TA (Hoyer, 2008).

The finding that rescuing neuronal OA and TA only partially restored aggression points to OA/TA being required either outside neurons or neuronal OA/TA being required at a specific (1) concentration, (2) time point, and (3) place to enable flies to express aggression. To test the importance of tyraminergic and octopaminergic neurons for the control of aggression, these neurons were selectively blocked. Inhibiting action potential generation via UAS-Kir2.1 expression (Baines, 2001) in Tdc2-neurons mimicked the TβhnM18 mutant phenotype. That is, Tdc2-GAL4/UAS-Kir2.1 males showed a significant decrease in lunges per meter compared to males both of the driver and of the effector line, with lunges occurring at a rate of about 22% of the controls. To restrict blockage of tyraminergic and octopaminergic neurons to a small time window, the temperature-sensitive UAS-shibire transgene was driven by Tdc2-GAL4. Blocking synaptic transmission only during the experimental period by raising the temperature to more than 30°C almost abolished aggression in Tdc2-GAL4/UAS-shits1 males compared to males of the same genotype fighting at the permissive temperature of 25°C. However, using the UAS-shits1 transgene for studying Drosophila aggression proved to be difficult due to a general trend of high temperature to reduce aggression. The general reduction in aggression due to high temperature made it difficult to detect differences between genotypes, especially when comparing UAS-shits1 males with Tdc2-GAL4/UAS-shits1 males at the high temperature, which required a manual evaluation. The marginal decrease in aggression found for UAS-shits1 males at 25°C compared to Tdc2-GAL4 males is presumably due to the slightly higher walking activity in UAS-shits1 males because the pure number of lunges was not affected. Despite the problems with using the UAS-shits1 transgene, the results obtained with both UAS-Kir2.1 and UAS-shits1 strengthen the hypothesis that octopaminergic neurons and potentially tyraminergic neurons are necessary for aggressive behavior (Hoyer, 2008).

In Drosophila as well as other arthropod species, OA is involved in modulating aggressive interactions. This study has taken various independent approaches all pointing at an important role of OA in this behavior. First, OA biosynthesis was genetically blocked at two steps in the metabolic pathway, resulting in strongly reduced male aggression. Then aggressive behavior was partially restored in one mutant by providing the missing metabolic enzyme in all cells via a transgene and in the other mutant by expressing the wild-type gene in octopaminergic and tyraminergic neurons. Finally, it was shown that aggression is suppressed when either action potential formation or synaptic transmission are blocked specifically in these neurons (Hoyer, 2008).

The first indication that OA might play a role in modulating Drosophila male aggression came from a study by Baier (2002), who observed in mutant TβhnM18 males a deficit in various aggressive behaviors when put together with control males. TβhnM18 males are, on average, 8% smaller than wild-type CS flies. According to the current data, this size difference alone would account for a substantial reduction in lunge frequency. Of even greater importance, their TβhnM18 flies also carried the white1118 mutation and therefore had white eyes, whereas their opponents were red eyed. The white1118 mutation by itself leads to a phenotype indistinguishable from the TβhnM18 mutation because both almost completely abolish aggression. In conclusion, Baier arrived at the right conclusion but, in retrospect, had no evidence (Hoyer, 2008).

In contrast to the current finding, Certel (2007) did not report a general decrease in aggression for TβhnM18 males when fighting against each other, presumably because the recording conditions used in their study and the current study were different. In the current setup, submissive males could not escape the small bottom area of the chamber and were, therefore, frequently attacked by the dominant male. It may be that this special enclosure situation, which led to high-lunge frequency in wild-type flies, reveals the impairment of the mutant (Hoyer, 2008).

Because TβhnM18 males have an ~10-fold increase in brain TA levels, the possibility was considered that excess TA might be the actual cause of reduced aggression. However, in Tdc2RO54 males lacking both neuronal OA and TA, aggression was as much reduced as seen in TβhnM18 males. Therefore, the aggression phenotype is attributed to low OA rather than high TA. Otherwise one would have to postulate that both high and low TA levels result in strongly reduced aggression. Immunohistochemical data indicate that in the fly's brain tyramine is not only localized in octopaminergic neurons but also in tyraminergic neurons specifically devoid of octopamine (Nagaya, 2002). Although these would be the best candidate neurons for mediating a presumed dose-dependent biphasic effect of TA, they would not show elevated TA levels in TβhnM18 mutant flies. In all, it is rather unlikely that TA has a major role in the suppression of aggression (Hoyer, 2008).

Expressing Tβh in all cells of adult TβhnM18 males via heat shock restored aggression to a small but significant number. Also expressing UAS-Tdc1 in Tdc2-neurons in Tdc2RO54 mutant males partially rescued aggression, indicating (1) that in both cases the defects were not caused by second-site mutations and (2) that some of the octopaminergic neurons in the brain are likely to mediate the effect. The latter argument is further strengthened by the finding that aggression is suppressed if these neurons are blocked. More specific GAL4 driver lines and manipulations of the dose and dynamics of OA in these neurons are needed to further elucidate its function in the control of aggression (Hoyer, 2008).

This study is based on an automated analysis of lunges, a single component of aggressive behavior in Drosophila males. Evaluating only a single indicator deals with aggression as if it were a unitary phenomenon and as if the various components were controlled by the same mechanism. This is unlikely to be true. As a starting point, this investigation was deliberately confined to this one aspect of aggression (Hoyer, 2008).

This study did not bring the recording and software analysis to perfection. Rather, it was decided to live with a low-tech setup and an error rate of about 11% that is mainly due to undetected lunges (tight exclusion criteria). The study was most severely troubled by the few false positives that prevented the detection of low rescue effects in mutants. For these cases, the lunge view software was developed, which allows the investigator to first loosen the criteria for lunges and to subsequently eliminate false positives. A second problem arose in the context of tussling, a high level aggressive behavior that consists of a mixture of boxing and lunging. During tussling sequences, lunges were less precisely detected. Fortunately, during the 15th to the 30th minute tussling was rare regarding all of the genotypes under investigation (Hoyer, 2008).

On the positive side, the automated counting of lunges allowed handling large amounts of data and guaranteed standardized evaluation. Because of its variance, a quantitative assessment of Drosophila aggression is exceedingly time consuming. The data reported here comprise a total of 480 hr of recording and a total of over 50,000 lunges. To fully analyze a clip, i.e., regarding the number of lunges, walking activity, the fly's body size, etc., the investigator needs to spend only ~3 min. Except for the very low end of the scale, the error rate is independent of lunge frequency. Fortunately, it is also largely independent of the genotypes used in this study (Hoyer, 2008).

In conclusion, In Drosophila, lunge frequency is strongly reduced without OA, but OA is apparently not necessary for triggering aggressive acts because flies lacking OA occasionally execute lunges. Consistently, crickets depleted of OA and dopamine still display aggression, but fights do not escalate to the same level as in controls, an effect that can be reversed by injecting the OA agonist CDM (Stevenson, 2005). Likewise, injecting one of the two OA receptor antagonists, epinastine or phentolamine, depresses aggression in crickets. Interestingly, the strength of the effect is context dependent. Whereas in naive crickets, only epinastine leads to a slight reduction in escalation level, the effect is stronger and seen for both antagonists if crickets are made to fly before the fight. Likewise in Drosophila, depletion of OA might affect aggression to varying degrees, depending on the situation. In the setup by Certel (2007), lack of OA results in no detectable effect, whereas it leads to a pronounced reduction in aggression when flies, as in the current setup, are forced to encounter each other at a high frequency. Thus, the strength of OA's influence on Drosophila aggression appears to be context dependent (Hoyer, 2008).

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 Gad1RNAi 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 d5HT1ARNAi knockdown in α' β' KCs nor Octβ2RRNAi 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., TβhRNAi, 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 TβhRNAi 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).

Octopamine indirectly affects proboscis extension response habituation in Drosophila melanogaster by controlling sucrose responsiveness

Octopamine is an important neurotransmitter in insects with multiple functions. This study investigated the role of this amine in a simple form of learning (habituation) in Drosophila. Specifically, it was asked if octopamine is necessary for normal habituation of a proboscis extension response (PER) to different sucrose concentrations. In addition, the relationship was analyzed between responsiveness to sucrose solutions applied to the tarsus and habituation of the proboscis extension response in the same individual. The Tyramine-beta-hydroxylase (Tβh) mutant lacks the enzyme catalyzing the final step of octopamine synthesis. This mutant was significantly less responsive to sucrose than controls. The reduced responsiveness directly led to faster habituation. Systemic application of octopamine or induction of octopamine synthesis by Tbetah expression in a cluster of octopaminergic neurons within the suboesophageal ganglion restored sucrose responsiveness and habituation of octopamine mutants to control level. Further analyses imply that the reduced sucrose responsiveness of Tβh mutants is related to a lower sucrose preference, probably due to a changed carbohydrate metabolism, since Tβh mutants survived significantly longer under starved conditions. These findings suggest a pivotal role for octopamine in regulating sucrose responsiveness in fruit flies. Further, octopamine indirectly influences non-associative learning and possibly associative appetitive learning by regulating the evaluation of the sweet component of a sucrose reward (Scheiner, 2014).

Modulation of Drosophila male behavioral choice

The reproductive and defensive behaviors that are initiated in response to specific sensory cues can provide insight into how choices are made between different social behaviors. This study manipulated both the activity and sex of a subset of neurons and found significant changes in male social behavior. Results from aggression assays indicate that the neuromodulator octopamine (OCT) is n for Drosophila males to coordinate sensory cue information presented by a second male and respond with the appropriate behavior: aggression rather than courtship. In competitive male courtship assays, males with no OCT or with low OCT levels do not adapt to changing sensory cues and court both males and females. A small subset of neurons was identified in the subesophageal ganglion region of the adult male brain that coexpress OCT and male forms of the neural sex determination factor, Fruitless (FruM). A single FruM-positive OCT neuron sends extensive bilateral arborizations to the subesophageal ganglion, the lateral accessory lobe, and possibly the posterior antennal lobe, suggesting a mechanism for integrating multiple sensory modalities. Furthermore, eliminating the expression of FruM by transformer expression in OCT/tyramine neurons changes the aggression versus courtship response behavior. These results provide insight into how complex social behaviors are coordinated in the nervous system and suggest a role for neuromodulators in the functioning of male-specific circuitry relating to behavioral choice (Certel, 2007).

To reduce or eliminate the function of OCT neurons, the Tyramine β-hydroxylase (Tβh) mutant lines were used (Monastirioti, 1996). The Tβh gene encodes the enzyme necessary to convert TYR to OCT, and null mutants (TβhnM18) produce no detectable OCT, whereas the hypomorphic TβhMF372 strain generates low levels of OCT. The revertant TβhM6 allele was used as the control. The original alleles were generated by P-element manipulations on the same chromosome. Subsequent manipulations were performed to replace the w1118 allele and backcrossed to Canton-S (CS) to maintain comparable genetic backgrounds. OCT, dopamine, and serotonin levels were verified in each Tβh allele by using HPLC (Certel, 2007).

Modulation of classical neurotransmitter action on target neurons adds great flexibility to synaptic output between neurons and is suggested to be at the core of important behavioral processes like learning and memory. In vertebrates, amines like serotonin, dopamine, and norepinephrine; peptides like arginine vasopressin, and oxytocin; gonadal steroids; and various glucocorticoids serve as well known neuromodulatory substances. Through selective actions at individual synaptic sites, neuromodulators coordinate the output of neuronal ensembles to generate behavioral patterns of varying complexity (Certel, 2007).

An elegant example of coordinating network output comes from studies with the stomatogastric ganglion of crustaceans. In this small neuronal ensemble, neuromodulators function either singly or in various combinations at multiple sites in the ganglion to alter the patterned output of the ganglion and thereby the movement of food through the stomach. An example of changing network ensembles in vertebrates is seen in studies of vole social behavior. Here, the distribution of oxytocin, vasopressin, and dopamine receptors within different brain regions appears linked to the differences seen in social behavior between prairie voles and montane voles (Certel, 2007 and references therein).

This paper focuses on the roles of octopamine, a phenolamine structurally related to the catecholamine norepinephrine, in modulating the choice between courtship and aggression in male flies. Norepinephrine has been shown to be important in many aspects of vertebrate behavior, including arousal, anxiety, learning and memory, opiate reward, and aggression. Among invertebrates, OCT influences foraging behavior in honey bees; resets aggressive motivation in crickets; and functions in appetitive associative learning, ethanol tolerance development, and possibly aggression levels in Drosophila. Like their vertebrate amine neuron counterparts, OCT neurons in Drosophila (1) are few in number but have enormous fields of innervation covering essentially all neuropil areas in the fly brain and (2) function by activating multiple G protein-coupled receptors (Certel, 2007).

Aggression and courtship usually are mutually exclusive behaviors. By examining the choices made between these behaviors by male flies, a powerful approach is offered with which to study the genetic and neural basis of complex behaviors. Multiple decision-making actions are required for each of these behaviors, including the processing of chemosensory and visual information and deciding whether another fly is a potential opponent or a potential mate. Using aggression and competitive courtship assays, OCT was found to be necessary for pairs of Drosophila males to respond to the sensory cues presented and to coordinate expression of the appropriate response: aggression. Feminizing OCT/tyramine (TYR) neurons in males also changes the aggression vs. courtship response behavior. Because the gene fruitless directs both courtship and aggression in flies, the expression patterns of OCT and the male forms of Fruitless (FruM) was analyzed and the were found to be coexpressed in distinct subesophageal ganglion (SOG) neurons in the male brain. This region receives the contact gustatory pheromone information thought to facilitate sex and species discrimination. The arborizations of one of the FruM-octopaminergic neurons were found to project bilaterally and appear to ramify in the posterior antennal lobe, multiple SOG layers, as well as the lateral accessory lobe (ventral body). These results offer insight into how sensory cues are integrated and modulated in the nervous system to direct sex-specific complex behaviors and indicate a role for the neuromodulator OCT in the functioning of the male-specific circuitry relating to behavioral choice (Certel, 2007).

Males and females react to environmental cues with distinct sex-specific innate behaviors particularly in the areas of courtship/reproduction and aggression/defense. Results from a number of studies have demonstrated that functional and structural sex differences in the brain can influence and direct these behaviors, but how sensory cues contribute to the appropriate response of one of these two mutually exclusive behaviors remains unclear. This study presents evidence that the neuromodulator OCT functions within a defined circuit to provide at least one means of regulating the choice between courtship and aggression. The results of these aggression studies indicate that male flies require OCT to respond with an appropriate aggressive response to another male. The results of the male-female courtship assays suggest that normal OCT function provides increased behavioral response confidence about the sensory cues being presented (Certel, 2007).

Identifying a potential mate or opponent relies on discriminating specific stimuli from background and then integrating this information with other sensory modalities. Anatomically, the extensive arrays of OCT-immunoreactive processes that are found throughout the Drosophila brain offer one such overlying integration network that may fine-tune sensory input and activate sex-specific behavioral subcircuits. In Drosophila, male-specific behavioral circuits are specified by the male-specific products of the fruitless gene. In this study, it was demonstrated that three VUM neurons in the male SOG coexpress FruM and OCT. The SOG is the primary taste-processing center in the fly. The sensory information sent to this neuropil includes the female pheromone recognition cues necessary for male courtship behavior. Therefore, an intriguing possibility is that OCT is necessary in the subset of FruM-positive SOG neurons to accurately relay contact gustatory pheromone information (Certel, 2007).

Morphological results suggest that a single neuron can provide a simple integration network of multisensory cues. The arborizations of one of the VUM 1 FruM-positive OCT neurons extensively ramify throughout multiple neuropil regions, including the SOG, posterior antennal lobe, and the lateral accessory lobe (ventral body), suggesting a link between various sensory modalities. Gustatory information from OCT/FruM SOG neurons could also be linked to higher-order processing centers through synaptic contacts with the male-specific SOG projections of FruM-expressing mAL neurons identified. The superior lateral protocerebrum has been proposed to be the output site of these interneurons. Linkages of this type may be of particular significance because FruM-expressing neurons play critical roles in two sex-specific social behaviors: aggression and courtship. Thus, the same circuits may need to integrate the context-specific sensory information necessary to direct the output of appropriate behavioral patterns (Certel, 2007).

How might OCT modify distinct SOG neurons to regulate behavioral choice by males? In the spider, OCT increases the overall sensitivity of mechanosensory neurons by local release from efferent endings. This local release suggests that sensory input from specific sensilla relative to others can be emphasized depending on behavioral circumstances. In the silkworm moth, OCT specifically increases the sensitivity of male pheromone-sensitive receptor neurons but not general odorant-sensitive responses. Recent modeling studies in vertebrates suggest that neuromodulators can play a key role at specific times in decision-making tasks by regulating competition between populations of neurons that represent choices. This regulation may allow an organism to integrate noisy sensory information and past experience to make optimal decisions (Certel, 2007).

Although the mouse neural pathways that mediate the output of two sex-specific behaviors, reproduction and defense, are anatomically segregated, a recent study identified a hypothalamic point of convergence that may function as a choice selection mechanism for sensory activation of defensive responses over reproduction. The results suggest that whether an individual male mouse responds with the appropriate behavior depends on the coordinated activation of the appropriate subcircuits by amygdalo-hypothalamic projections. Likewise the different behavioral outputs of Drosophila males and females could be generated through the activation of sex-specific segregated neural ensembles. However, behavioral differences also could emerge through sex-specific modulation of circuits that are common to both sexes. In males, FruM proteins are expressed in small groups of neurons throughout the CNS, and eliminating FruM expression in a neuronal subset has profound effects on the progression of male courtship behaviors. At the gross level almost all of the FruM-producing neurons have counterparts in the female and in terms of function, a recent report indicates that the sex-specific reproductive behaviors of females and males involve shared neural circuits. The splicing of fruM-specific transcripts have been proposed to modify neurons common in both sexes for male-specific functions through differences in neuron morphology and/or physiology (Certel, 2007).

In addition to changing the activity of OCT neurons, OCT/TYR neurons were feminized in an otherwise masculine brain and altered male behavioral choice was demonstrated. The results from OCT immunostaining do not indicate any sex-dependent changes in SOG neuron number. The identification of a sex-independent marker for the FruM-positive OCT neurons should allow determination of whether feminizing these neurons changes either their branching patterns, their synaptic connections, or their OCT-related biochemical properties. Further examination of these OCT/FruM SOG neurons should offer a behaviorally relevant ensemble with which to address questions of sex-specific morphology and function-related physiology (Certel, 2007).

A subset of octopaminergic neurons are important for Drosophila aggression

Aggression is an innate behavior that is important for animal survival and evolution. This study examined the molecular and cellular mechanisms underlying aggression in Drosophila. Reduction of the neurotransmitter octopamine, the insect equivalent of norepinephrine, decreased aggression in both males and females. Mutants lacking octopamine did not initiate fighting and did not fight other flies, although they still provoked other flies to fight themselves. Mutant males lost to the wild-type males in fighting and in competing for copulation with females. Enhanced octopaminergic signaling increased aggression in socially grouped flies, but not in socially isolated flies. Genetic rescue experiments were carried that revealed the functional importance of neuronal octopamine and identified a small subset of octopaminergic neurons in the subesophageal ganglion as being important for aggression (Zhou, 2008).

The assay for aggression contains a food pad in the middle of a closed chamber. The small size of the chamber increases the number of chances for contact and also makes it possible to videotape six chambers simultaneously. Two flies were placed into each chamber and videotaped. Analysis was carried out for the first 10 min after both flies were in the chamber. Latency of fighting was measured by the time from the placement of the flies into the chamber to the first fight; frequency was measured by the number of fights in a specific pattern in the first 10 min. Only fighting patterns involving physical engagement were scored in these studies and included lunging, holding, boxing and tussling. Holding, boxing and tussling are collectively shown as HIF (high-intensity fighting) (Zhou, 2008).

It has been reported that aggression is reduced in flies that carry mutations in both the white gene and the Tβh gene, which encodes an important enzyme for octopamine synthesis. A critical test was missing to distinguish whether the phenotype was the result of the white mutation or the TβhnM18 mutation. A previous study examined the fighting behavior of white mutants and observed an almost complete impairment in aggressive behavior of w1118 null mutants. Consistent with these results, it was found that the white mutants were defective in aggression, as evidenced by lengthened fighting latency and reduced frequency of lunging and HIF. When white mutants were paired with wild-type flies, fights were initiated and won more by the wild-type flies than by the white mutants. These results indicate that white mutations affect aggression in the absence of mutations in Tβh and that the role of octopamine in aggression could not be established by experiments with flies carrying mutations for both white and Tβh (Zhou, 2008).

To investigate the role of octopamine, TβhnM18 mutant flies were tested that had no mutation at the white locus. Octopamine was completely depleted in TβhnM18 mutants. Fighting latency was significantly prolonged in TβhnM18 mutants, whereas lunging and holding frequencies were reduced in TβhnM18 mutants. The frequency of boxing and tussling was very low in the wild-type flies. Although TβhnM18 mutants had a decreased frequency of boxing and tussling, it was not significantly different from wild type (Zhou, 2008).

In Drosophila, both the levels and the patterns of aggression are different between males and females. The most prominent pattern in female aggression is head butting, which is similar to lunging in males, except that females charge the opponents with the head rather than forelegs. Whether octopamine was also involved in female aggressive behavior was tested. Pairs of females of the same genotype were tested. Fighting latency was prolonged in TβhnM18 mutant females, whereas head butting frequency was reduced, indicating a common function of octopamine in male and female aggression (Zhou, 2008).

Octopamine level is reduced in TβhnM18 mutants throughout development and adult life. To test whether octopamine is involved in aggressive behavior in adults, the activity of octopaminergic neurons was manipulated by using Tdc2-Gal4 to drive the expression of shibirets (shits), a temperature-sensitive mutant of dynamin that can inhibit the vesicle recycling, thus blocking neural transmissions at the restrictive temperature. Neither Tdc2-Gal4 nor UAS-shits alone affected aggression at 23°C or 31°C. When both Tdc2-Gal4 and UAS-shits were present, aggression was reduced within 15 min of shifting to the restrictive temperature. Latency was longer at 31°C than that at 23°C, and frequencies of lunging and HIF were lower at 31°C. Consistent with previous results, the finding that acutely silencing octopaminergic neurons phenocopies the TβhnM18 mutant indicates that the aggression phenotype in TβhnM18 mutants is not a result of developmental defects (Zhou, 2008).

Reduced aggression between the TβhnM18 mutants could mean that a mutant fly either does not initiate aggression against another fly or does not elicit aggression by others. To distinguish between these possibilities, TβhnM18 mutants were paired with wild-type flies. Wild-type flies still fought the TβhnM18 mutants, whereas the mutants did not initiate fighting and did not fight back. These results indicate that the mutants do not fight, but are still able to elicit fighting by others (Zhou, 2008).

Aggression is important for the resource holding power of mammals. In flies, a study with six wild-type males found a correlation between aggressiveness and mating success, but ebony males were found to have reduced mating with enhanced territorial aggression. To determine whether and how the resource holding power was affected in TβhnM18 mutants, their ability to compete for females and food was tested. When placed in the fighting chamber with food in the middle, wild-type flies won the majority of fights. When a mutant male and a wild-type male were placed in the same chamber with a wild-type virgin female, the chance for copulation was significantly lower for the TβhnM18 mutant than for the wild type. The total time on the flies spent on the central patch of food in the fighting chamber was measured as the occupancy duration. TβhnM18 mutants spent significantly less time than the wild-type flies on the food pad. These results indicate that TβhnM18 mutants are less successful than wild-type in their competition for resources and for females (Zhou, 2008).

The decreased aggression of TβhnM18 could result from a general defect in movement, an inability of male flies to find their usual opponents or a defect in motivation for any action. To test whether the aggression phenotype of TβhnM18 is an indirect result of other behavioral defects, locomotion was assessed by measuring the speed of individual flies in a round chamber. No difference was found between TβhnM18 mutants and wild-type flies. TβhnM18 mutants were then tested for their ability to sense and avoid specific odorants. The avoidance index of TβhnM18 mutants to benzaldehyde did not differ significantly from that of the wild type, indicating that there was no defect in the odor sensitivity and the avoidance behavior in TβhnM18 mutants (Zhou, 2008).

To test the ability of flies to distinguish between males and females, a decapitated wild-type female and a decapitated wild-type male were placed in two opposite ends of a chamber that contained a TβhnM18 mutant or wild-type male. TβhnM18 males were similar to the wild-type males in their preference for spending more time with the females, showing little interest in the males, which suggests that reduced aggression in TβhnM18 mutants was not a result of an inability to distinguish the sexual identities of opponents (Zhou, 2008).

Whether courtship behavior was affected in TβhnM18 mutants was tested. In the typical male-female courtship assay, TβhnM18 and wild-type males had similar courtship indices, initiation latencies and mating latencies. Reduced aggression between males could also be a consequence of increased male-male courtship behavior. When tested with a male-male courtship assay, TβhnM18 males were similar to wild-type males in that neither showed significant male-male courtship (Zhou, 2008).

It has recently been reported that octopamine is required for making a choice between aggression and courtship, a conclusion that is based on an analysis of the behavioral patterns of TβhnM18 mutants after a male fly showed wing extension. In contrast, a general decrease of aggression, but no significant increase of courtship in TβhnM18 mutants was found. The frequency of unilateral wing vibration, which is an early step in courtship, was indistinguishable between TβhnM18 mutants and wild types, whereas the frequency of wing threat (bilateral wing extension), a typical step in aggression, was significantly reduced in TβhnM18 mutants. It is possible that the previous study might have mixed wing extensions of different kinds (such as the unilateral wing vibration and bilateral wing threat) and interpreted a simple reduction of aggression as a concomitant reduction of aggression and increase of courtship, or, alternatively, different setups for measuring aggression might have contributed to the discrepancies of behavior outputs (Zhou, 2008).

The results suggest that the aggression phenotype of TβhnM18 mutants is not secondary to defects in general behavior patterns (Zhou, 2008).

Social experience is important for the development of aggressive behavior in both mammals and insects, with socially isolated males fighting more than those that were raised in groups. In these studies, male flies were raised in isolation or in groups of two or ten flies of the same age until day 5. Flies with identical rearing conditions were paired and tested. Grouping markedly reduced aggression. Furthermore, it was found that a group of two flies was as effective as a group of ten flies at reducing aggression (Zhou, 2008).

To address the question of whether enhancing octopamine signaling could restore aggressiveness in socially grouped flies, the effects of chlorodimeform (CDM) on fly aggression were tested. Notably, treatment of grouped flies with CDM, an octopamine agonist, reduced the fighting latency and increased the lunging frequency without significantly affecting the HIF frequency. Moreover, TβH was overexpressed in grouped flies by a TβH transgene under the control of the heat shock promoter (hspTβh. After heat shock induction for 30 min, flies were allowed to recover for 3 h. hspTβh flies had shortened fighting latency, increased lunging frequency and increased HIF frequency. Heat shock for the same amount of time did not affect aggression in wild-type controls (Zhou, 2008).

The UAS-NaChBac/Tub-Gal80ts system was used to activate octopaminergic neurons and examined its effect on aggression. At the permissive temperature, Gal80ts binds to and inhibits the transcription activation activity of Gal4. When shifted to the restrictive temperature, Gal80ts becomes nonfunctional, allowing Gal4 to activate the transcription of NaChBac, a bacterially derived voltage-sensitive sodium channel with a lower threshold for activation and slower kinetics for inactivation compared with voltage-sensitive sodium channels in flies, which causes neuronal activation in Drosophila. The tubulin (Tub) αTub84b promoter drives the expression of Gal80ts in all cells. Combining UAS-NaChBac and Tub-Gal80ts with Tdc2-Gal4 allowed expression of NaChBac in adult octopaminergic neurons, thus activating them post-developmentally. Tdc2-Gal4, UAS-NaChBac or Tub-Gal80ts alone did not affect aggression. When all three components were present, aggression in grouped flies was increased after shifting to the restrictive temperature, with shorter latency and higher lunging and HIF frequencies, indicating that the activation of octopaminergic neurons reversed the reduction of aggression by social grouping of flies (Zhou, 2008).

It was also asked whether enhancing octopamine signaling could promote aggression in socially naive flies. No significant effect of either octopamine agonist chlordimeform treatment or overexpression of TβH by heat shock-inducible promoter was detected in flies that were raised in social isolation. Two possibilities can explain this phenomenon. The first possibility is that octopamine is involved in resetting aggression after social experience. The second possibility is that the level of aggressiveness is saturated in socially naive flies, which occludes further enhancement of aggression by other treatments. The first possibility would be further supported if the octopamine concentration is changed by social experience. Thus the concentrations of bioamines in the brains of males was examined by high-performance liquid chromatography with electrochemical detection, but no difference was detected in octopamine level between grouped and isolated flies. This does not completely rule out the first possibility, since the concentration of octopamine in a limited number of neurons could be changed, but this could not be detected when whole heads were assayed (Zhou, 2008).

Octopamine exists both inside and outside of the nervous system, functioning as either a neurotransmitter or a hormone in insects. It is therefore important to investigate whether the aggression phenotype of Tβh mutants is a result of defects in the nervous system (Zhou, 2008).

In Drosophila, two enzymes, dTdc1 and dTdc2, are involved in the first step of octopamine synthesis, with dTdc1 being expressed outside of the brain and dTdc2 being expressed in the brain. The expression pattern of Tdc2-Gal4 and Tdc1-Gal4 was confirmed by staining the adult male brains of Tdc2-Gal4/UAS-mCD8:GFP and Tdc1-Gal4/UAS-mCD8:GFP with antibody to Tβh. GFP expression was not detected in the brain of Tdc1-Gal4/UAS-mCD8:GFP heterozygous flies. In Tdc2-Gal4/UAS-mCD8:GFP flies, only two populations of octopaminergic neurons, one in the antenna lobe and another in the SOG, were positive for both GFP and TβH immunoreactivity. Tdc2-Gal4 was unable to drive expression in octopaminergic neurons in other brain regions, such as the protocerebrum, the fan-shaped body and the central complex (Zhou, 2008).

To functionally differentiate the neuronal and endocrine contribution of octopamine, Tdc2-Gal4 and Tdc1-Gal4 were used to drive UAS-Tβh in TβhnM18 mutant males. Tdc2-Gal4, Tdc1-Gal4 or UAS-Tβh alone did not affect aggression in TβhnM18 mutant males. The combination of Tdc2-Gal4 and UAS-Tβh rescued aggression deficiency in TβhnM18 males: the latency was shortenedand the frequencies for both lunging and HIF were increased. Tβh expression, driven by Tdc2-Gal4, could also effectively rescue the aggression phenotype of female TβhnM18 mutants. In contrast, Tdc1-Gal4-driven TβH expression failed to rescue the aggression phenotype of TβhnM18 mutants with regard to fighting latency, lunging frequency and HIF frequency, arguing against the involvement of hormonal octopamine in aggression (Zhou, 2008).

Other Gal4 lines were used to express TβH in different brain regions of TβhnM18 mutants in an attempt to find the location of the subset of octopaminergic neurons involved in aggression. The TβhnM18 mutant phenotype could not be rescued by TβH expression under the control of either c309-Gal4, which drives expression in the mushroom bodies, or MJ286-Gal4, which drives expression in a cluster of neurons in lateral protocerebrum. Therefore, it is concluded that the aggression phenotype in TβhnM18 mutants is the result of a TβH deficiency in the nervous system (Zhou, 2008).

To further define the subset of octopaminergic neurons that is involved in aggression, use was made of the drivers Cha-Gal4 and Cha-Gal80. These drivers were made by fusing Gal4 or Gal80 to the promoter of choline acetyltransferase (Cha), and they drive expression in cholinergic neurons (Zhou, 2008).

Since it has been shown that the antennal lobe and the SOG populations of octopaminergic neurons positive for Tdc2-Gal4 are involved in aggression, it was checked whether these neurons overlapped with neurons in which Cha-Gal4 could drive gene expression. All of the antennal lobe and most of the SOG neurons that were immunoreactive to antibody to TβH were also positive for GFP driven by Cha-Gal4. Behaviorally, it was found that Cha-Gal4-driven TβH expression could not rescue the aggression phenotype in TβhnM18 mutants. These results suggest that neurons that were positive for TβH and Cha-Gal4 are not responsible for the aggression deficit in TβhnM18 mutants (Zhou, 2008).

Cha-Gal80 allowed the complementary experiment to be carried out: asking the importance of neurons that are positive for octopamine, but negative for Cha-driven gene expression. The Tdc2-Gal4 driver was used to express GFP in the presence of Cha-Gal80, and it was found that GFP expression in all of the antennal lobe neurons and most of the SOG neurons was suppressed, with only 2-5 SOG octopaminergic neurons being GFP positive. Therefore, the combination of Tdc2-Gal4 and Cha-Gal80 could be used to drive TβH expression in those SOG neurons. It was found that TβH expression that was driven by Tdc2-Gal4 and Cha-Gal80 could rescue the aggression phenotype in TβhnM18 mutants. These results indicate that a distinct subset of octopaminergic neurons in the SOG is functionally important for aggression (Zhou, 2008).

The results support the notion that the aggression phenotype in Tβh mutants was not a secondary result of changes in sexual discrimination or sexual behavior. The results have shown that Tβh mutants display normal sexually related activities. The conclusion of a recent report of octopamine involvement in making a choice between aggression and courtship (Certel, 2007) is not supported by the current results, probably because different experimental setups were used or because the previous report did not separately analyze unilateral wing extension (a part of courtship) and bilateral wing extension (the wing threat in aggression), indicating that a simple reduction of aggression might have thus been interpreted as an increase in courtship. When unilateral and bilateral wing extension were separately counted,an expected decrease of bilateral wing extension was indeed found in Tβh mutants, but found no change in unilateral wing extension were found. Consistent with these results, a previous study also observed generally decreased aggression behavior in Tβh mutants, although the role of octopamine in courtship was not examined (Zhou, 2008).

Octopamine is the insect counterpart of norepinephrine and its receptors in insects are homologous to mammalian adrenoceptors. The best evidence for norepinephrine involvement in aggression was provided by mice that lacked the gene for the α2c-adrenergic receptor, an autoinhibitory receptor. Mice without this receptor display increased aggressive behavior, whereas α2c overexpression decreased aggression. In Dbh knockout mice completely lacking noradrenaline, the aggressive response is essentially eliminated in a resident-intruder protocol. One important question in the future should be whether and how norepinephrine and its receptors in defined locations of the mammalian brain are involved in aggression (Zhou, 2008).

Octopamine regulates sleep in Drosophila through protein kinase A-dependent mechanisms

Sleep is a fundamental process, but its regulation and function are still not well understood. The Drosophila model for sleep provides a powerful system to address the genetic and molecular mechanisms underlying sleep and wakefulness. This study shows that a Drosophila biogenic amine, octopamine, is a potent wake-promoting signal. Mutations in the octopamine biosynthesis pathway produced a phenotype of increased sleep, which was restored to wild-type levels by pharmacological treatment with octopamine. Moreover, electrical silencing of octopamine-producing cells decreased wakefulness, whereas excitation of these neurons promoted wakefulness. Because protein kinase A (PKA) is a putative target of octopamine signaling and is also implicated in Drosophila sleep, its role in the effects of octopamine on sleep was investigated. Decreased PKA activity in neurons rendered flies insensitive to the wake-promoting effects of octopamine. However, this effect of PKA was not exerted in the mushroom bodies, a site previously associated with PKA action on sleep. These studies identify a novel pathway that regulates sleep in Drosophila (Crocker, 2008).

By modulating the excitability of octopamine-producing cells, the output of these cells was manipulated. In mammals, one can record from specific cell populations to determine when the cells fire action potentials. Although this assay is difficult to do in flies, it was possible to electrically modulate the cells through expression of K+ and Na+ ion channels. When octopamine-producing cells were more depolarized (expression of a Na+ channel), the animal was awake more and unable to stay asleep, whereas when the cells were hyperpolarized (expression of a K+ channel), the animals slept more (Crocker, 2008).

Based primarily on larval crawling assays, octopamine and tyramine were implicated previously in locomotor behavior (O'Dell, 1994; Gong, 2004; Saraswati, 2004; Scholz, 2005). Specifically, larvae move slower through quadrants when they have decreased octopamine levels (the TβHnm18 and Tdc2RO54 mutants). More recent work showed that adult Tdc2RO54 flies also have a decrease in locomotor activity attributable to the lack of tyramine (Hardie, 2007). The data showing differences in activity in the Tdc2RO54 and the TβHnm18 mutants support the claim that tyramine plays an important role in locomotion. Thus, whereas increased levels of tyramine in Tbh mutants increase activity, decreased levels in Tdc mutants decrease locomotor activity. However, both mutations increase sleep, which is most likely attributable to the loss of octopamine. In addition to overall sleep, it was found that other sleep parameters such as latency to sleep and arousal threshold are affected in flies carrying these mutations. It is inferred that tyramine plays a role in locomotion, but octopamine specifically affects arousal states (Crocker, 2008).

Studies of other invertebrate species support a role for octopamine in arousal (Corbet, 1991). In fact, octopamine agonists are potential natural pesticides because they cause insect species to 'walk off' the leaves (Roeder, 1999). As in Drosophila, changes in octopamine levels affect behavior in honey bees, as demonstrated through feeding and injection of octopamine as well as through analysis of endogenous levels of octopamine. Fussnecker (2006) showed that injections of octopamine promote flying in honeybees. In addition, octopamine and tyramine regulate other behaviors in honeybees such as hive maintenance and foraging (Schulz, 1999; Schulz, 2001; Wagener-Hulme, 1999; Barron, 2002). Octopamine and tyramine also modulate sensory input in honeybees (Kloppenburg, 1995; Scheiner, 2002). In the locust, octopamine mediates heightened arousal in response to new visual stimuli (Bacon, 1995). Bacon found that a specific subset of octopamine-producing neurons in the brain of the locust fires during the presentation of new visual stimuli, causing dishabituation of the descending contralateral movement detector interneuron. Interestingly, application of endogenous octopamine can mimic this state of heightened arousal. This study suggests that octopamine serves to promote arousal in Drosophila. It is possible that the increased arousal seen with too much octopamine, or decreased arousal with too little, is a result of improper gating of sensory stimuli, but without electrophysiological data it is not possible to draw any conclusions. Note also that the Tdc2 cells important for sleep and arousal in the fly brain have not been identified yet (Crocker, 2008).

Previous studies, octopamine was fed to flies to rescue or verify a phenotype of the TβHnm18 flies. The ability of octopamine to rescue egg laying in TβHnm18 mutants was assayed in this manner, because TβHnm18 flies are unable to release eggs. Animals were placed on different levels of octopamine, and 10 mg/ml octopamine over a period of 6 d provided maximal rescue (Monastirioti, 1996). Using the same concentration, this study found that a steady increase in octopamine levels led to a decrease in nighttime sleep. Based on the specific effect on nighttime sleep, it is speculated that octopamine levels are already high during the daytime, thereby precluding any effects of an increase. This analysis is supported by the Na+ channel data in which a significant decrease in total sleep was found only during the nighttime sleep periods. It is speculated that, normally, activity of these cells is low at night, and so expression of the Na+ channel causes them to fire more and release octopamine at an abnormal time, thereby producing a decrease in sleep. Similar results, indicating nighttime-specific effects, were obtained with overexpression of Tdc2. Work in other insects also supports the idea of modulated octopamine release. Pribbenow (1996) demonstrated that honeybees who are already in a heightened arousal state of antennae scanning do not change scanning frequency in response to octopamine administration, but, in animals scanning at a low frequency, injections of octopamine significantly increase scanning (Crocker, 2008).

Thse data suggest that the effects of octopamine are mediated through PKA-dependent signaling. In mammals, there are nine different adrenergic receptors, some of which signal through PKA. The α1 adrenergic receptor is the only receptor associated with a wake-promoting effect in that the agonist methoxamine causes an increase in waking . However, the antagonist has no effect on total sleep. It is important to note that the α1 receptor in mammals is thought to be coupled to phospholipase C and Gq. The β adrenergic receptors (which are coupled to cAMP and PKA) probably do not have specific effects on sleep in mammals because, contrary to known effects of norepinephrine, the agonist increases sleep and the antagonist decreases sleep. Studies in Drosophila may be better able to identify biogenic amine receptors relevant for sleep because of the ease of genetic manipulation. Many G-protein-coupled receptors in Drosophila display activity that allows their bona fide classification as octopamine receptors. The current data suggest that receptors sensitive to mianserin are likely to be involved in regulating fly sleep. Because mianserin inhibits cAMP signaling, these data not only further support a role for PKA but also implicate β receptors in octopamine action. It is noted that none of these receptors is known to display a circadian cycling profile (Crocker, 2008).

Given that PKA has been shown to regulate sleep in Drosophila, a link between the various molecules that affect Drosophila sleep is starting to be apparent. Interestingly, however, octopamine does not appear to act through the mushroom bodies, a structure known to mediate effects of PKA on sleep and also to express a class of octopamine receptors. Because flies lacking mushroob bodies still have substantial amounts of sleep, it is clear that other parts of the fly brain can drive sleep. The current study shows that even PKA can affect sleep in regions outside the mushroom body. Defining the site of action of sleep-regulating molecules such as octopamine should help to identify these other brain regions (Crocker, 2008).

Trace amines differentially regulate adult locomotor activity, cocaine sensitivity, and female fertility in Drosophila melanogaster

The trace biogenic amines tyramine and octopamine are found in the nervous systems of animals ranging in complexity from nematodes to mammals. In insects such as Drosophila melanogaster, the trace amine octopamine is a well-established neuromodulator that mediates a diverse range of physiological processes, but an independent role for tyramine is less clear. Tyramine is synthesized from tyrosine by the enzyme tyrosine decarboxylase (TDC). The identification of two Tdc genes in has been reported Drosophila: the peripherally-expressed Tdc1 and the neurally-expressed Tdc2. To further clarify the neural functions of the trace amines in Drosophila, normal and cocaine-induced locomotor activity were examined in flies that lack both neural tyramine and octopamine because of mutation in Tdc2 (Tdc2RO54). Tdc2RO54 flies have dramatically reduced basal locomotor activity levels and are hypersensitive to an initial dose of cocaine. Tdc2-targeted expression of the constitutively active inward rectifying potassium channel Kir2.1 replicates these phenotypes, and Tdc2-driven expression of Tdc1 rescues the phenotypes. However, flies that contain no measurable neural octopamine and an excess of tyramine due to a null mutation in the tyramine d-hydroxylase gene (TβHnM18) exhibit normal locomotor activity and cocaine responses in spite of showing female sterility due to loss of octopamine. The ability of elevated levels of neural tyramine in TβHnM18 flies to supplant the role of octopamine in adult locomotor and cocaine-induced behaviors, but not in functions related to female fertility, indicates mechanistic differences in the roles of trace amines in these processes (Hardie, 2007).

To more clearly define behavioral roles for the trace amines tyramine and octopamine, adult locomotor activity and cocaine-induced behaviors were examined in flies that lack one or both of these amines. Behavioral analyses show that Tdc2RO54 flies, which lack both tyramine and octopamine, have dramatically reduced basal activity levels and are hypersensitive to an initial dose of cocaine. Though these locomotor phenotypes are most severe in flies homozygous for the Tdc2RO54 mutation, they are also evident in Tdc2RO54 heterozygotes and in flies expressing the Tdc2-Kir2.1 transgenes. The cocaine phenotype observed in Tdc2RO54 flies is the most extreme example of hypersensitivity observed to date. In contrast to prior expectation, Tdc2RO54 flies show normal sensitization to repeated cocaine exposures, which precludes involvement of neural tyramine or octopamine in this process. This conclusion was critically based on a linkage between the inactive gene (iav) and levels of brain tyramine/octopamine, a linkage that is now tenuous. TβHnM18 flies, which lack octopamine and have increased levels of neural tyramine, show normal responses to cocaine and normal levels of locomotion. The simplest interpretation of thes data would indicate that tyramine, not octopamine, is involved in the regulation of normal and cocaine-induced locomotor activity in adult flies, since normal behaviors are observed in the TβHnM18 flies that are totally deficient in neural octopamine. However, it is possible that the elevated levels of tyramine present in these flies can supplant the function of octopamine, since tyramine can activate the octopamine receptor DmOA1A if present at elevated concentrations (Balfanz, 2005). By this mechanism, either octopamine or tyramine, at sufficiently high levels such as in TβHnM18, would act to supress the hypersensitivity to cocaine and locomotor inactivity phenotypes observed in the absence of either amine. In C. elegans, Alkema (2005) report results analogous to those above, in that Tdc mutant animals show more severe behavioral and egg laying phenotypes than animals mutant for TBH. They reach the conclusion that tyramine has specific behavioral effects based on the ability to identify putative tyramine specific neurons that are Tdc immunoreactive but do not express TBH-GFP. Ablation of these neurons has distinct behavioral consequences that they attribute to tyramine. This study has not been able to identify a subset of specific tyramine-containing neurons in Drosophila, thus limiting independent manipulation of this amine. In an attempt to further isolate the phenotypic effects of tyramine vs octopamine, behavioral rescue of the Tdc2RO54 flies was attempted by feeding tyramine or octopamine. Feeding of these trace amines readily rescues the female sterility of Tdc2RO54 (Cole, 2005); however, feeding proved ineffective in rescuing the hypersensitivity to cocaine or the locomotor inactivity in Tdc2RO54, even when doses were adjusted to yield near-normal levels of brain amines and when feeding was continued for the entirety of post-embryonic development. Intriguingly, these studies did not indicate that the ectopic tyramine was converted into octopamine, as if the ectopically supplied amines were not transported into the appropriate neurons where the metabolic conversion could take place. This latter finding is consistent with failure to find a candidate octopamine/tyramine reuptake transporter in the fly genome (Hardie, 2007).

Trace amines can function via interactions with the presynaptic dopamine transporter (DAT), where they can stimulate dopamine efflux, or via interactions with specific G protein coupled receptors known as trace amine receptors (TAARs) (reviewed in Roeder, 2005). In vertebrates, there is data indicating that both mechanisms play a role in mediating the effects of trace amines, but functional in vivo evidence is only available for the latter: TA-stimulated behavioral effects are observed in DAT knockout mice, suggesting that in the absence of functional DAT activity, stimulation of specific TAAR's provides a mechanism for the action of trace amines (Sotnikova, 2004; Sotnikova, 2005). Both mechanisms are also possible in Drosophila, since amine efflux via dDAT is stimulated by tyramine (Porzgen, 2001), and several receptors for tyramine and octopamine have been detected in the fly central nervous system (reviewed in Roeder, 2005). For trace amine functions in Drosophila ovulation, it is now clear that octopamine acts via the OAMB receptor, since an OAMB mutant parallels the ovulation deficient phenotype of flies lacking octopamine (Han, 1998; Lee, 2003). For the motor functions studied in this report, however, it is not yet possible to make a clear discrimination of mechanism. There is abundant evidence that biogenic amines, including the trace amines, play a modulatory role in the control of motor behaviors in the nervous systems of both vertebrates and invertebrates. In humans, the importance of dopamine in motor control is most evident in Parkinson’s disease, where degeneration of dopamine cell bodies in the substantia nigra results in movement disorders. In arthropod species, injections of dopamine, serotonin, or octopamine into the central nervous system evoke distinct motor postures; similarly, in Drosophila, octopamine and dopamine elicit strong locomotor activity in decapitated preparations of the nerve cord (Yellman, 1997). Further, complementary pharmacological and genetic data indicate that synaptically released dopamine is an important stimulator of arousal threshold and locomotor activity in Drosophila. Thus, if tyramine or octopamine function by altering dopamine signaling, the reduced locomotor activity in Tdc2RO54 would indicate that tyramine is a positive mediator of dopamine signaling and release. It seems highly likely that the primary mediators of cocaine responses in Drosophila are the amine transporters dDAT and dSERT; however, evidence as to which is more important in these responses is ambiguous. Cocaine binds the Drosophila serotonin transporter dSERT more tightly than the Drosophila dopamine transporter dDAT, and flies expressing gene products expected to decrease dopamine and serotonin synthesis and release are somewhat paradoxically hypersensitive to cocaine, apparently because of developmental compensation. Short term feeding of flies with an inhibitor of dopamine synthesis, in contrast, leads to reduced sensitivity to cocaine. Therefore, tyramine could potentially be interacting with dopamine and serotonin signaling pathways (Hardie, 2007).

Whatever the precise mechanism, the data show a striking difference in terms of tyramine/octopamine functions in adult locomotor behaviors relative to functions in ovulation. First, TβHnM18 flies are female sterile and show defects in ovulation, whereas these flies show normal locomotor and cocaine-induced behaviors. Second, female fertility, but not cocaine responsiveness, is readily rescued by feeding of octopamine and to a lesser extent, tyramine. Thus, a plausible model to account for the failure to rescue the adult behavioral phenotypes of Tdc2RO54 by tyramine/octopamine feeding would posit a need for focal release of trace amines from tyramine/octopamine CNS neurons. In contrast, trace amine feeding can readily rescue ovarian neuromuscular functions (Cole, 2005), potentially via non-focal release mechanisms since trace-amine neuronal endings are reported to be at some distance from sites of function (Lee, 2003). This is also consistent with the ability of octopamine to modulate muscular contractions of denervated ovary preparations (Middleton, 2006; Rodriguez-Valentin, 2006; Hardie, 2007 and references therein).

There is preliminary evidence that trace amines are involved in the modulation of vertebrate locomotor activity. The trace amine receptor TAAR1 is present in the CNS of rodents and primates and is stimulated by tyramine, b-phenylethylamine and amphetamine, all of which are locomotor activators. However, mice lacking TAAR1 have normal locomotor activity, which could be due to stimulation of the remaining TAAR receptors. Mice lacking TAAR1 are hypersensitive to the stimulatory effects of the psychostimulants amphetamine and cocaine and respond to a single dose with significantly increased dopamine and norepinephrine release in the dorsal striatum relative to wild type controls. These results indicate a striking conservation of the effects of trace amines on psychostimulant responses between Drosophila and mice. Despite a significant divergence in the synthesis pathways of trace amines between vertebrates and invertebrates, this evidence indicates that the overall role of trace amines may remain at least partially conserved throughout evolution (Hardie, 2007).

Flight initiation and maintenance deficits in flies with genetically altered biogenic amine levels

Insect flight is one of the fastest, most intense and most energy-demanding motor behaviors. It is modulated on multiple levels by the biogenic amine octopamine. Within the CNS, octopamine acts directly on the flight central pattern generator, and it affects motivational states. In the periphery, octopamine sensitizes sensory receptors, alters muscle contraction kinetics, and enhances flight muscle glycolysis. This study addresses the roles for octopamine and its precursor tyramine in flight behavior by genetic and pharmacological manipulation in Drosophila. Octopamine is not the natural signal for flight initiation because flies lacking octopamine [tyramine-β-hydroxylase (TβH) null mutants] can fly. However, they show profound differences with respect to flight initiation and flight maintenance compared with wild-type controls. The morphology, kinematics, and development of the flight machinery are not impaired in TβH mutants because wing-beat frequencies and amplitudes, flight muscle structure, and overall dendritic structure of flight motoneurons are unaffected in TβH mutants. Accordingly, the flight behavior phenotypes can be rescued acutely in adult flies. Flight deficits are rescued by substituting octopamine but also by blocking the receptors for tyramine, which is enriched in TβH mutants. Conversely, ablating all neurons containing octopamine or tyramine phenocopies TβH mutants. Therefore, both octopamine and tyramine systems are simultaneously involved in regulating flight initiation and maintenance. Different sets of rescue experiments indicate different sites of action for both amines. These findings are consistent with a complex system of multiple amines orchestrating the control of motor behaviors on multiple levels rather than single amines eliciting single behaviors (Brembs, 2007).

How are rhythmical motor behaviors initiated, maintained, and terminated? For many years, neuroscientists have debated whether motor behaviors were produced by chains of reflexes or by intrinsically oscillating central networks. Pioneering work on locust flight set the stage for today's well accepted concept of central pattern generation by demonstrating that rhythmic motor output could be induced by nonrhythmical stimulation of the nerve cord without sensory feedback. The underlying networks are central pattern generators (CPGs), which are found at the heart of motor networks in all animals (Brembs, 2007 and references therein).

Neuromodulators play a major role in activating and modifying CPG activity (Marder, 2001). The central release of specific neuromodulators or mixtures of different modulators can initiate distinct motor patterns (Nusbaum, 2001). Pioneering studies in locusts have demonstrated that microinjection of the biogenic amine octopamine (OA) into distinct neuropil regions elicits either walking or flight motor patterns in isolated ventral nerve cords (Sombati, 1984). This has led to the 'orchestration hypothesis' (Hoyle, 1985) assuming that neuromodulator release into specific neuropils configures distinct neural assemblies to produce coordinated network activity. Monoamines have also been assigned to aggression, motivation, and mood in vertebrates and invertebrates (Baier, 2002; Kravitz, 2003; Stevenson, 2005; Popova, 2006). Furthermore, specific cognitive functions have been assigned to monoamine codes, such as that in flies OA mediates appetitive learning but dopamine mediates aversive learning (Schwaerzel, 2003; Riemensperger, 2005). In mammals, dysfunctions in monoamine neurotransmission are implicated in neurological disorders, including Parkinson's disease, schizophrenia, anxiety, and depression (Kobayashi, 2001; Taylor, 2005; Brembs, 2007 and references therein).

However, recent work from areas as diverse as Parkinson's disease (Scholtissen, 2006) and Drosophila larval motor behavior suggests that the chemical codes producing specific motor behavior outputs are bouquets of different amines rather than single ones (Saraswati, 2004; Fox, 2006). This study tests this hypothesis by genetic and pharmacological dissection of flight behavior in Drosophila. For >20 years, OA has been assigned as the sole modulator controlling insect flight. In contrast, this study demonstrates that flight is controlled by the combined action of OA and tyramine (TA). OA and TA are decarboxylation products of the amino acid tyrosine, with TA as the biological precursor of OA. In insect flight systems, OA assumes a variety of physiological roles affecting central neuron excitability (Ramirez, 1991), synaptic transmission (Evans, 1979; Leitch, 2003), sensory sensitivity (Matheson, 1997), hormone release (Orchard, 1993), and muscle metabolism (Mentel, 2003). Almost every organ is equipped with OA receptors (Roeder, 1999). TA receptors have been cloned recently in many insect species (Blenau, 2003), and physiological functions for TA have been demonstrated (McClung, 1999; Nagaya, 2002). This paper discusses the multiple possible levels of OA and TA action on Drosophila flight behavior (Brembs, 2007).

Flies lacking OA and having increased TA levels (TßH null mutants) show a profound decrease in flight initiation and maintenance compared with wild-type controls. Five lines of evidence suggest that morphology, kinematics, and development of the flight machinery are not impaired in TßH mutants: (1) wing-beat frequencies, (2) wing-beat amplitudes, (3) flight muscle structure (length of myofibrils), and (4) the number and overall dendritic structure of flight motoneurons are unaffected in TßH mutants, and (5) the behavioral phenotype can acutely be rescued in adult flies. Although acute application of OA is sufficient to elicit flight in a number of different insect preparations, OA is not necessary for the initiation of flight in Drosophila but modulates flight initiation and maintenance. Even flies without any OA/TA-containing neurons can fly. Therefore, OA is either not a necessary natural signal for flight initiation or Drosophila flight initiation is a unique case (Brembs, 2007).

A novel finding is that flies lacking OA and with tyramine receptors (TARs) blocked show wild-type-like flight behavior. It is important to note that the TßH phenotype comprises OA knock-out plus eightfold increased TA levels. Pharmacological blockade of TARs yields the most efficient rescue of the TßH mutants, even outscoring replacement of OA by heat-shock plus TAR blockade. However, blocking TARs in wild-type flies does not increase flight initiation or maintenance. This indicates that TA inhibits flight behavior only at abnormally high TA levels. Furthermore, with regard to flight maintenance, the inhibitory effects of TA take place only at low OA levels, because OA replacement without affecting the TA system also yields rescues of the initial and the average flight bout durations. In contrast, the responsiveness to stimulation is rescued best by blocking TA. Therefore, flight initiation is most likely inhibited by high TA levels, regardless of the OA levels. Accordingly, feeding TßH mutants OA does not rescue flight initiation but restoring tyramine-ß-hydroxylase activity by heat shock does, because only the latter manipulation decreases the levels of TA by conversion of TA into OA. Therefore, the most parsimonious interpretation is that OA is necessary for flight maintenance, and TA acts most likely as an inhibitor, especially for flight initiation at high concentrations (Brembs, 2007).

This interpretation is further supported by ablating all OA/TA neurons by expressing the apoptosis factor reaper in these cells. Flies without OA/TA neurons show the same massive changes in flight behavior as TßH mutants. Therefore, genetic ablation of all TA/OA-containing neurons does not phenocopy genetic ablation of the OA-producing enzyme paired with pharmacological block of TA action. How can these seemingly contradictory results be explained? Clearly, the pharmacological treatment with yohimbine is effective; it fully rescues the mutant phenotype. The ablation of the OA/TA neurons is equally effective, ruling out methodological flaws. However, yohimbine does most likely not block all TA action, whereas genetic ablation of all TA-containing neurons does. Thus, the action of TA presumably follows a bell-shaped curve, with its presence necessary for normal flight but hindering flight initiation and maintenance at high concentration. OA is required most likely for flight maintenance because feeding it to TßH mutants fully rescues normal flight maintenance. However, OA supplementation in the food might also exert rescuing effects in TßH mutants by downregulating TA via feedback inhibition. In summary, the most compelling explanation for the data are that OA is boosting flight maintenance, low levels of TA are required for flight maintenance and initiation, and inhibitory TA actions fall in place at high TA and low OA levels (Brembs, 2007).

The finding that OA and TA are involved in regulating flight emphasizes the role of TA as an independent neurotransmitter in invertebrates. Further supporting this role, tyramine-like immunoreactivity has been demonstrated in non-octopaminergic cells of Caenorhabditis elegans and locusts. Moreover, at least one Drosophila amine receptor is specific for TA and does not cross-react with OA (Cazzamali, 2005). Furthermore, OA and TA receptor distributions in the insect CNS differ considerably from each other. Functionally, exogenous TA increases chloride conductances in Drosophila malphigian tubules, alters body wall muscle excitatory junction potentials, and can rescue cocaine sensitization in Drosophila. In mammals, the physiological roles for trace amines such as TA and OA are mostly unknown, but they have been implicated in a variety of neurological disorders, and receptors specific for TA have been identified. In invertebrates, a role of endogenous TA as an important transmitter/modulator has been shown for Drosophila locomotor and olfactory avoidance behavior, as well as for C. elegans motor behavior (Brembs, 2007 and references therein).

Previous studies suggested that OA acts as a potent, direct stimulator of flight muscle metabolism (Wegener, 1996; Mentel, 2003). Accordingly, it was expected that especially prolonged flight would be affected in TßH mutants, attributable to insufficient fuel supply. In contrast, all flight parameters are similarly affected in TßH mutants. The initial flight bout duration is decreased ~40 times, and the total flight duration is decreased ~30 times in TßH mutants. Moreover, flight behavior changes in TßH mutants are rescued by blocking TA action alone, leaving OA levels unaltered. This is hard to reconcile with direct effects of OA on flight metabolism and would require independent effects of OA and TA on flight metabolism. These considerations render metabolism unlikely as the site of action for OA. Therefore, amine effects on Drosophila flight initiation and maintenance are more likely to be mediated by effects on the nervous system (Brembs, 2007).

Two main OA/TA effects on flight behavior can be observed: maintenance of flight and the probability of initiating flight. In principle, both could be controlled by aminergic action on the CPG and/or on the fly's sensory system. It is well established that OA acts on the central pattern generators in a number of insect species, but central actions of TA are not known. OA has also been reported to increase the responsiveness of flight-associated sensory cells in insects, and TA could conceivably reduce excitability of sensory neurons as Drosophila TARs activate chloride currents (Brembs, 2007 and references therein).

OA and TA have been implicated as agonist and antagonist, respectively, controlling locomotor behavior in Drosophila larvae (Saraswati, 2004; Fox, 2006) and in C. elegans (Alkema, 2005). This raises the possibility of a general, opponent OA/TA control of locomotor behavior in invertebrates. The current results make it unlikely that OA and TA simply act antagonistically on the same targets because, with regard to flight initiation and maintenance, OA and TA probably have different sites of action and TA effects are important only at high TA and low OA levels. Nevertheless, in some preliminary experiments, whether TßHnM18 mutant adults show also walking behavior deficits was tested. Neither the overall motor activity per unit time nor the number of walking bouts differed between wild-type and TßHnM18 mutant flies. However, a slight but statistically significant reduction was found in walking speed in TßHnM18 mutants. These findings indicate that aminergic modulation by OA and TA does not act generally on locomotor performance but specifically affects different aspects of motor behaviors (Brembs, 2007).

In summary, the emerging picture is that, for some motor behaviors, the concerted interaction of specific biogenic amines is more important than the concentration of single amines. The current study is the first to suggest that the antagonistic actions of OA and TA are not a general feature of all invertebrate locomotor behaviors but specifically affect distinct aspects of different motor behaviors. It provides evidence that OA and TA do not simply act antagonistically on the same targets but most likely mediate their effects on motor performance by affecting different targets in a dose-dependent manner. The next steps toward understanding amine function for motor behavior is to determine their sites of action during behavior. One possibility addressing this question is to combine pharmacological and genetic rescues and test immunocytochemically where the OA and TA levels are restored in which rescue procedure, how behavior is affected in these different manipulations, and where the various subtypes of TA and OA receptors are localized. Ultimately, a complete understanding of the mechanism by which various modulators interact on different parts of the brain and other tissues to control motor behavior will require a large number of targeted manipulations of each individual circuit component separately (Brembs, 2007).

Dopamine and Octopamine regulate 20-hydroxyecdysone level in vivo in Drosophila

The effects of increased level of dopamine (DA) (feeding flies with DA precursor, L-dihydroxyphenylalanine, L-DOPA) on the level of 20-hydroxyecdysone (20E) and on juvenile hormone (JH) metabolism in young (2-day-old) wild type females (the strain wt) of Drosophila virilis have been studied. Feeding the flies with L-DOPA increased DA content by a factor of 2.5, and led to a considerable increase in 20E level and a decrease of JH degradation (an increase in JH level). The levels of 20E were measured in the young (1-day-old) octopamineless females of the strain TβhnM18 and in wild type females, Canton S, of Drosophila melanogaster. The absence of OA led to a considerable decrease in 20E level (earlier it was shown that in the TβhnM18 females, JH degradation was sharply increased). This paper studied the effects of JH application on 20E level in 2-day-old wt females of D. virilis. It was demonstrated that an increase in JH titre results in a steep increase of 20E level. The supposition that biogenic amines act as intermediary between JH and 20E in the control of Drosophila reproduction is discussed (Rauschenbach, 2007).

Levels of JH degradation have been studied in females with a twofold increase of the DA con tent. The D. melanogaster strains ste and ebony carry a mutation that drastically decreases activity of the enzymes converting DA into N-β-alanyldopamine. Young females of both strains have considerably lower JH degradation levels and the mature flies have higher levels compared to wild type, Canton S. It has also been showm that feeding flies of wt strain of D. virilis with DA results in a decrease in JH degradation in young (nonovipositing 2-day-old) females, while in the mature (7-day-old) ones, it led to an increase in JH degradation (Rauschenbach, 2007 and references therein).

The results obtained in the present study when measuring JH degradation levels in 2-day-old L-DOPA-fed wt females agree with the above data: an increase in DA content leads to a decrease in JH degradation level, which is inferred to be indicative of an increase in JH level (Rauschenbach, 2007).

In wild type females of D. melanogaster, the regulation of JH synthesis and degradation tends to be opposing: both JH titre and JH synthesis in young (1-day-old) wild type D. melanogaster females were substantially higher than in mature (5-6-day-old) flies. At the same time, JH degradation in young wild type D. melanogaster females is significantly lower than in the mature ones. Females of the mutant apterous of D. melanogaster were shown to have dramatically decreased JH synthesis and sharply increased JH degradation. Considering all the above, it has been suggested that (1) JH synthesis and degradation are under a common control system in the adult females of Drosophila, and (2) the factors stimulating the hormone synthesis inhibit its degradation and vice versa. This notion agrees well with the fact that an experimental increase of the JH titre in wt females of D. virilis leads to a decrease in its degradation. The idea of the correlated regulation of JH synthesis and degradation in insects is also supported by data showing that ovariectomy of Acheta domesticus females results in the simultaneous decrease of JH synthesis and increase in the activity of JH-esterase that degrades the hormone. In microarray experiments, it has been shown that treatment of D. melanogaster starved females with JH leads to a down-regulation of JH-epoxide hydrolase 3 (the main JH-hydrolizing enzyme in adults females of D. melanogaster) (Rauschenbach, 2007).

It has been shown that an increase in 20E level in young wt D. virilis females leads to an increase in DA content, and in sexually mature ones, to its decrease. In that case and if there is a feedback regulation (a direct effect of DA on 20E metabolic system), an increase in DA content in young females should result in a decrease in 20E level. Data presented in this study indicate that this is not the case: the 20E level is increased in young females with an increased DA content. At the same time, a rise in JH level (a decrease of its degradation) produced in young Drosophila females by the increase in DA content should lead to a rise of 20E because JH activates ecdysone synthesis in ovaries of young females. Data in this study correlate with this: in JH-treated wt females, the 20E level is dramatically increased. Thus, it is proposed that DA has an effect on 20E metabolism, but this effect is indirect and mediated through the JH metabolic system. Levels of JH degradation have been studied in females of D. melanogaster octopamineless strain (Gruntenko, 2000). Both young and mature octopamineless females have JH degradation levels much higher (JH levels much lower) than those in wild type, Canton S, flies. If OA, like DA, regulates 20E through the JH metabolic system, one could expect octopamineless females to have 20E level lower than in wild type. The data suggest that this is the case. The supposition that OA regulates 20E level indirectly via the JH metabolic system agrees with the results of the experiment in which OA content was increased by feeding wt females of D. virilis with the amine (Gruntenko, 2007): JH degradation decreased (JH level went up) and 20E level increased in the OA-treated females (Rauschenbach, 2007)..

Summarizing the results of the present study, the following scheme is proposed of the reciprocal regulation of biogenic amines and gonadotropins in Drosophila. DA increases JH level (inhibits JH degradation and apparently stimulates synthesis) in young females and decreases it (stimulates degradation and apparently inhibits synthesis) in sexually mature flies. There is a feedback in this regulation; a rise in JH level leads to a decrease in DA content in young females and its rise in the mature ones. OA leads to a rise of JH level (inhibits JH degradation and, evidently, stimulates its synthesis) in young and mature females. 20E regulates JH indirectly via the DA metabolic system; a rise in 20E level increases DA content in young and decreases it in mature females, thus leading to a de- crease of JH degradation (a rise in its titre) in both. DA influences 20E level indirectly via the JH metabolic system. OA is also likely to regulate 20E indirectly via the JH metabolic system (Rauschenbach, 2007).

Waking experience affects sleep need in Drosophila: Experience-dependent changes in sleep need require dopaminergic modulation, cAMP signaling, and a particular subset of long-term memory genes

Sleep is a vital, evolutionarily conserved phenomenon, whose function is unclear. Although mounting evidence supports a role for sleep in the consolidation of memories, until now, a molecular connection between sleep, plasticity, and memory formation has been difficult to demonstrate. Drosophila as a model to investigate this relation; the intensity and/or complexity of prior social experience stably modifies sleep need and architecture. Furthermore, this experience-dependent plasticity in sleep need is subserved by the dopaminergic and adenosine 3',5'-monophosphate signaling pathways and a particular subset of 17 long-term memory genes (Ganguly-Fitzgerald, 2006).

Sleep is critical for survival, as observed in the human, mouse, and fruit fly, and yet, its function remains unclear. Although studies suggest that sleep may play a role in the processing of information acquired while awake, a direct molecular link between waking experience, plasticity, and sleep has not been demonstrated. Advantage was taken of Drosophila genetics and the behavioral and physiological similarities between fruit fly and mammalian sleep to investigate the molecular connection between experience, sleep, and memory (Ganguly-Fitzgerald, 2006).

Drosophila is uniquely suited for exploring the relation between sleep and plasticity for at least two reasons. (1) Fruit flies sleep. This is evidenced by consolidated periods of quiescence associated with reduced responsiveness to external stimuli and homeostatic regulation -- the increased need for sleep that follows sleep deprivation. (2) Drosophila has been successfully used to elucidate conserved mechanisms of plasticity. For example, exposure to enriched environments, including the social environment, affects the number of synapses and the size of regions involved in information processing in vertebrates and Drosophila. In the fruit fly, these structural changes occur in response to experiential information received within a week of emergence from pupal cases. Although brain plasticity is not limited to this period, the first week of emergence does coincide with the development of complex behaviors in Drosophila, including sleep. Hence, daytime sleep, which accounts for about 40% of total sleep in adults, is highest immediately after eclosion and stabilizes to adult levels 4 days after emergence (Ganguly-Fitzgerald, 2006).

To assess the impact of waking experience during this period of brain and behavioral development, individuals from the wild-type C-S strain were exposed to either social enrichment or impoverishment immediately at eclosion and were tested individually for sleep 5 days later. Socially enriched individuals (E), exposed to a group of 30 or more males and females (1:1 sex ratio) before being tested, slept significantly more than their socially impoverished (I) siblings, who were housed individually. This difference in sleep [DeltaSleep (E)] was restricted to daytime sleep. Socially enriched individuals consolidated their daytime sleep into longer bouts of ~60 min compared with their isolated siblings, who slept in 15-min bouts. In contrast, nighttime sleep was unaffected by prior social experience, corresponding with observations that daytime sleep is more sensitive to sex, age, genotype, and environment, when compared with nighttime sleep. This effect of social experience on sleep persisted over a period of days. Moreover, it was a stable phenotype: When socially enriched, longer-sleeping individuals and socially impoverished, shorter-sleeping siblings were sleep-deprived for 24 hours, they defended their respective predeprivation baseline sleep quotas by returning to these levels after a normal homeostatic response (Ganguly-Fitzgerald, 2006).

Experience-dependent modifications in sleep have long been observed in humans, rats, mice, and cats. But what is the nature of the experiential information that modifies sleep need in genetically identical Drosophila? Differences in sleep need in socially enriched and socially impoverished individuals were not a function of the space to which they were exposed -- flies reared in 2-cc tubes slept the same as those reared in 40-cc vials. Neither did it arise out of differences in reproductive state or sexual activity between the two groups: Socially impoverished mated and virgin individuals slept the same, as did socially enriched individuals from mixed-sex or single-sex groups. Further, differences in sleep were not a reflection of differences in overall activity (measured as infrared beam breaks) between the two groups. Although social context can reset biological rhythms, mutations in clock (Clkjerk), timeless (tim01), and cycle (cyc01) disrupt circadian rhythms but had no effect on experience-dependent responses in sleep need (Ganguly-Fitzgerald, 2006).

Because social interaction requires sensory input, fly strains that were selectively impaired in vision, olfaction, and hearing were evaluated . Blind norpA homozygotes failed to display a response in sleep to waking experience: Sleep need in norpA mutants did not increase after exposure to social enrichment. In contrast, norpA/+ heterozygotes with restored visual acuity slept more when previously socially enriched. Attenuating visual signals by rearing wild-type (C-S) flies in darkness also abolished the effect of waking experience on sleep. Compromising the sense of smell while retaining visual acuity also blocked experience-dependent changes in sleep need: Socially enriched smellblind1 mutants slept the same as their impoverished siblings. As confirmation, neurons carrying olfactory input to the brain were specifically silenced [Or83b-Gal4/UAS-TNT, and it was observed that sleep in these flies was also not affected by prior waking experience. Auditory cues, however, did not affect the relation between experience and sleep. Finally, sleep need in individual Drosophila increased with the size of the social group to which they were previously exposed. Socially isolated flies slept the least, whereas those exposed to social groups of 4, 10, 20, 60, and 100 (1:1 sex ratio) showed proportionately increased daytime sleep need. When rendered blind, however, flies did not display this relation between sleep need and the intensity of prior social interactions (Ganguly-Fitzgerald, 2006).

If sensory stimulation received during a critical period of juvenile development directs the maturation of the adult sleep homeostat, then subsequent environmental exposure should not affect adult sleep time and consolidation. Alternatively, if experience-dependent modifications in sleep are a reflection of ongoing plastic processes, this phenomenon would persist in the adult. It was observed that sleep in flies was modified by their most recent social experience regardless of juvenile experience. Shorter sleeping socially impoverished adults became longer sleepers when exposed to social enrichment before being assayed. Conversely, longer sleeping socially enriched flies became shorter sleepers after exposure to a period of social isolation. Moreover, repeated switching of exposure between the two social environments consistently modified sleep, reflecting an individual's most recent experience (Ganguly-Fitzgerald, 2006).

An estimation of neurotransmitter levels in whole brains revealed that short-sleeping, socially impoverished individuals contained one-third as much dopamine as their longer-sleeping, socially stimulated isogenic siblings. Silencing or ablating the dopaminergic circuit in the brain [TH-Gal4/UAS-TNT and TH-Gal4/UAS-Rpr specifically abolished response to social impoverishment in individuals that were reared in social enrichment. Similar results were obtained when endogenous dopamine levels were aberrantly increased, by disrupting the monoamine catabolic enzyme, arylalkylamine N-acetyltransferase, in Datlo mutants. Hence, abnormal up- or down-regulation of the dopaminergic system prevented behavioral plasticity in longer sleeping, socially enriched individuals when switched to social impoverishment (Ganguly-Fitzgerald, 2006).

The observation that dopaminergic transmission affects experience-dependent plasticity in sleep need is particularly compelling, given its role as a modulator of memory. Mutations in 49 genes implicated in various stages of learning and memory were screened to assess their impact on experience-dependent changes in sleep need. Of these, only mutations in short- and long-term memory genes affected experience-dependent plasticity in sleep need. Mutations in dunce (dnc1) and rutabaga (rut2080) have opposite effects on intracellular levels of adenosine 3',5'-monophosphate (cAMP), but are both correlated with short-term memory loss. In dnc1 mutants, waking experience had no impact on subsequent sleep need. This effect was partially rescued in dnc1/+ heterozygotes, but complete rescue was only achieved when a fully functional dunce transgene was introduced into the null mutant background. rut2080, however, selectively abolished the ability of socially enriched adults to demonstrate decreases in sleep after exposure to social impoverishment, which was reminiscent of aberrant dopaminergic modulation. Similarly, of the long-term memory genes screened, 17 (~40%) specifically disrupted the change in sleep need in socially enriched adults after exposure to social impoverishment. For example, overexpression of the Drosophila CREB gene repressor, dCREB-b, resulted in socially enriched flies that continued to be longer sleepers even after exposure to social impoverishment. As a control, overexpression of the dCREB-a activator yielded wild-type phenotypic read out. It is noteworthy that not all long-term memory mutants had a disrupted relation between experience and sleep. Instead, the particular subset of genes identified, only half of which are expressed in the mushroom bodies, may specifically contribute to pathways that underlie sleep-dependent consolidation of memories (Ganguly-Fitzgerald, 2006).

Finally, to assess the correlation between sleep and memory, male flies trained for a courtship conditioning task that generated long-term memories were measured for sleep after training. Males whose courtship attempts are thwarted by nonreceptive, recently mated females or by males expressing aphrodisiac pheromones form long-term associative memories as evidenced by subsequently reduced courtship of a receptive virgin female. Trained males that formed long-term memories slept significantly more than their untrained siblings and wake controls (ones that were sleep-deprived while the experimental flies were being trained). Exposure to a virgin female did not alter sleep need. As before, this increase in sleep was associated with longer daytime sleep bouts in trained individuals compared with controls. Further, sleep deprivation for 4 hours immediately after training abolished training-induced changes in sleep-bout duration, as well as courtship memory. Although these results are intriguing, invertebrate memory is particularly sensitive to extinction by mechanical perturbations. However, gentle handling that ensured wakefulness, but not mechanical stimulation, immediately following training, also abolished subsequent courtship memory. Furthermore, sleep deprivation per se did not affect the formation of long-term memory: Trained flies that were allowed to sleep unperturbed for 24 hours and then subjected to 4 hours of sleep deprivation retained courtship memory (Ganguly-Fitzgerald, 2006).

In summary, this study has demonstrate a rapid and dynamic relation between prior social experience and sleep need in Drosophila. In particular, experience-dependent changes in sleep need require dopaminergic modulation, cAMP signaling, and a particular subset of long-term memory genes, supporting the hypothesis that sleep and neuronal activity may be inexorably intertwined. These observations are compelling given two recent studies have demonstrating a central role of the mushroom bodies in sleep regulation and emphasize the importance of establishing Drosophila as a model system to investigate the molecular pathways underlying sleep and plasticity (Ganguly-Fitzgerald, 2006).

Dopamine and octopamine differentiate between aversive and appetitive olfactory memories in Drosophila

The catecholamines play a major role in the regulation of behavior. This study investigated the role of dopamine and octopamine (the presumed arthropod homolog of norepinephrine) during the formation of appetitive and aversive olfactory memories. For the formation of both types of memories, cAMP signaling is necessary and sufficient within the same subpopulation of mushroom-body intrinsic neurons. In contrast, memory formation can be distinguished by the requirement for different catecholamines, dopamine for aversive and octopamine for appetitive conditioning. These results suggest that in associative conditioning, different memories are formed of the same odor under different circumstances, and that they are linked to the respective motivational systems by their specific modulatory pathways (Schwaerzel, 2003; full text of article).

In the honeybee, OA mediates at least some of the reinforcing capacity of sugar reward in an associative olfactory discrimination task (Hammer, 1998; Menzel, 1999). Therefore, the role of OA in sugar and electric shock learning was investigated in Drosophila using the TβH-deficient mutant, TβHM18. The biosynthetic pathway to OA is blocked in this mutant, and it has no detectable levels of OA (Monastirioti, 1996). When tested for electric shock memory, mutant TβHM18 flies performed like the wild-type controls, but when tested for sugar memory, the mutant was severely impaired, showing a performance index near zero. Thus, blocking OA synthesis does not cause a general learning deficit but specifically interferes with sugar learning. This phenotype could be rescued by a transgene containing the wild-type TβH cDNA downstream of the hsp70 promoter. With heat shock, these flies (TβHM18; hsp70-TβH HS+) showed wild-type performance in sugar memory. The heat shock itself had no memory-enhancing effect in mutant TβHM18 flies, supporting the implicit assumption that OA levels can be rescued by restoring enzymatic function (Schwaerzel, 2003).

Besides lacking OA, the mutant TβHM18 accumulates tyramine, its direct precursor (Monastirioti, 1996) and a functional neurotransmitter (Nagaya, 2002). To test whether the increase in tyramine or the absence of OA causes the phenotype, TβHM18 mutant flies were fed OA (10 mg/ml) for either 1 or 18 hr before training. OA-fed mutant TβHM18 flies performed like wild-type flies. Surprisingly, a feeding period of as little as 1 hr was sufficient to restore the learning-memory defect, indicating that OA is taken up by the neurons via a rapid mechanism. This is in line with several reports in bees, in which the feeding of OA increases levels of this neurotransmitter in the brain and behavioral effects occur within the range of minutes after uptake (Schwaerzel, 2003).

To distinguish between an effect of OA during acquisition and retrieval, OA was fed to the mutant flies just after training, and memory was tested 1 hr later. No rescue of performance was found in these flies, whereas in control flies the same OA feeding regimen had no deleterious effect. Therefore, it is concluded that OA is required during acquisition. Whether it is also needed during retrieval cannot be decided (Schwaerzel, 2003).

The results support three major conclusions: (1) during the association of an olfactory cue with either a sugar reward or an electric shock punishment, both forms of olfactory memories require cAMP signaling within the same 700 Kenyon cells of the MBs; (2) for memory retrieval but not acquisition with either of the two reinforcers, output from this same set of cells is required. Hence, the memory must be formed and stored upstream of this synaptic level. (3) Sugar and electric shock reinforcement are mediated by different modulatory neurotransmitters, DA in case of electric shock and octopamine (OA) in case of sugar reward. These findings confirm and extend previous work, concluding that output synapses of Kenyon cells are the site of olfactory memory. Appetitive and aversive olfactory memories are localized to the same neuropil Associative behavioral adaptations are mediated by the plasticity of synapses within neural circuits. But what are the smallest units of memory? Do they correspond to the modulation of a single synapse or to the concerted change of many or all synapses in a circuit? Attempts to localize olfactory memory in the Drosophila brain have provided partial answers to these questions (Schwaerzel, 2003).

In many species, including Aplysia, mouse, and Drosophila, the type-1 AC has been shown to be critical in synaptic plasticity. No cases of cAMP-independent associative synaptic or behavioral plasticity have yet been reported conclusively. In Drosophila, one of the corresponding mutants, rut, shows abnormal performance in every learning paradigm tested so far. By identifying the minimally sufficient set of neurons that in a rut mutant brain need to express a wild-type form of the RUT protein to restore a particular memory performance, one can localize the memory trace of the corresponding behavioral adaptation. This approach was successfully applied to two types of memory in Drosophila, heat box memory and olfactory memory (Schwaerzel, 2003).

Using the same approach in a side-by-side comparison between sugar and electric shock reinforcement, the current results show that wild-type rut-AC expression in 700 Kenyon cells (25%-30% of total) rescues memory performance for both kinds of reinforcement. Thus, aversive and appetitive olfactory memories are both mediated by synaptic plasticity in the same group of cells (Schwaerzel, 2003).

Attributing the rescue to an effect on synaptic plasticity in the adult Kenyon cells disregards the possibility that the genetic manipulation might rescue a developmental function of rut-AC, necessary later in the adult for memory. Several lines of evidence argue for an adult function, but only recently has a new genetic manipulation been designed that definitely rules out a developmental effect. Use of a temperature-sensitive Gal80, a suppressor of Gal4, ensured that wild-type rut-AC was expressed only during adulthood (Schwaerzel, 2003).

Although the current experiments do not specify where in the Kenyon cells cAMP signaling is required, the existing evidence suggests a presynaptic mechanism at Kenyon cell output synapses. At the Drosophila larval neuromuscular junction, cAMP signaling is presynaptically involved in plasticity. For the sensory-motor synapses mediating classical conditioning of the gill withdrawal reflex in Aplysia, it has been firmly established that the cAMP cascade is involved presynaptically. No conclusive example of a postsynaptic contribution of cAMP signaling has been reported (Schwaerzel, 2003).

In the Aplysia synapses, plasticity has a postsynaptic component based on a mechanism resembling the NMDA receptor and long-term potentiation in mammals. In Drosophila olfactory conditioning, a similar postsynaptic contribution is unlikely to play a role during the first 3 hr, because this effect would require neurotransmitter release from the presynapse, which can be blocked during acquisition and memory retention without a deleterious effect on memory, using shits1, a conditional blocker of synaptic transmission (Schwaerzel, 2003).

The tentative presynaptic effect of cAMP signaling locates the synaptic plasticity underlying olfactory memory to the synapses connecting Kenyon cells to MB output neurons. These are found in the MB lobes, including the rostral peduncle and spur. Additional support for cAMP signaling to occur in the lobes rather than calyx is derived from the 'memory' gene amn, which has been shown to be involved in cAMP regulation. The putative AMN neuropeptide is required exclusively in two prominent neurons, the so-called dorsal paired medial neurons, that profusely innervate the MB lobes. Other components of the cAMP pathway such as rut and receptors for DA and OA, all are predominantly expressed in the adult MB lobes (Schwaerzel, 2003).

Associative synaptic plasticity depends on the convergence between impulses from two signals, the CS and the US. Considering the proposed role of rut-AC as a molecular coincidence detector, one can assume that the MB input neurons carrying the US for sugar and electric shock should also connect to the lobes, although their direct anatomical identification is pending (Schwaerzel, 2003).

The current results show that acquisition of an olfactory memory with electric shock is dependent on the dopaminergic system, whereas acquisition with sugar depends on the octopaminergic system. OA as neurotransmitter in sugar learning seems to be conserved between Drosophila and the honeybee. The bee VUMmx1 neuron, an unpaired neuron localized in the subesophageal ganglion, appears to be octopaminergic and has been shown to carry some of the reinforcing properties of the US. It innervates the calices, antennal lobes, and lateral protocerebrum but not the MB lobes. Nevertheless, the learning paradigms used [individual conditioning of the proboscis extension reflex in bees vs the population-based conditioned osmotaxis in Drosophila] are different; therefore, it might be too early to compare the sugar memories in the bee and Drosophila with respect to its organization on a circuit level. Unfortunately, the role of the monoamines in aversive conditioning has not been tested in bees (Schwaerzel, 2003).

These findings raise the question of whether the effects of the two catecholamines on electric shock and sugar learning can be generalized to other appetitive and aversive reinforcers and to positive and negative behavioral modulation in general. In the monkey, midbrain dopaminergic neurons have been described that carry the reinforcing properties of a US in appetitive but not aversive conditioning. It will be interesting to see whether a similar dissociation between modulatory systems for appetitive and aversive conditioning, with the contingency between good-bad and monoamines exchanged, also applies to the monkey, and, potentially, to humans (Schwaerzel, 2003).

Separate memory traces for electric shock and sugar conditioning had been suggested previously, because these have different kinetics of consolidation and decay. The distinct effects of the two catecholamines in the reinforcement pathways discovered in this study underline this notion. Surprisingly, however, localization experiments assign the two memories to the same neuropil structure, a subset of 700 Kenyon cells (Schwaerzel, 2003).

Based on the functional anatomy of the olfactory pathway, odors are assumed to be represented in the MBs by specific sets of Kenyon cells. For an odorant to become predictive of a given reinforcing event (e.g., sugar or electric shock), the output synapses of this set of Kenyon cells should be modified to drive an MB output neuron mediating the conditioned response (e.g., approach or avoidance). MB input neurons representing the appropriate reinforcers (e.g., sugar or electric shock) should provide the modulatory input. At present it is still not known whether the identified monoamines, OA and DA, are the modulatory neurotransmitters at the site of synaptic plasticity or act further upstream in the respective US pathway. The former alternative is supported by the observation that the MB lobes are equipped with DA and OA receptors that can couple to AC of the rut type via Gs protein. In addition, the neurons relevant for electric shock learning send TH-Gal4-positive fibers to the MB neuropil at the level of the spur and the vertical lobes (Schwaerzel, 2003).

The respective output neurons are prespecified to announce sugar or electric shock and so are the modulatory neurons. Hence, these form specific pairs (US-CR pairs) that are functionally linked to adapt the CR neuron to one of many odors in the conditioning events. Two schemes can be proposed of how the US-CR pairs and sets of Kenyon cells might be interconnected. The two diagrams differ with respect to the organization of odor representations in the MBs. If the same odors were represented by several sets of Kenyon cells, each set could be connected with just one US-CR pair. In this case, sugar and electric shock memories could be formed in different sets, both specifically responding to the same odorant, but one being modulated by OA, the other by DA. Both these sets would be contained within the set of 700 Kenyon cells of the 247-Gal4 driver line. Alternatively, if each odor is represented by only one set, the Kenyon cells should be responsive to multiple modulatory inputs. In this case, both memories would be formed within the same cells. The synapses of the US-CR pairs with the Kenyon cells should be closely associated, and these synaptic domains would have to be independently modulated by cAMP signaling. Because Drosophila can associate many events (US) with odors, Kenyon cells may accommodate many such US-CR pairs. A requirement for space at the Kenyon cells may then explain the stalk-like structure of MBs. At present it is not possible to distinguish between these two alternatives (Schwaerzel, 2003).

Distinct octopamine cell population residing in the CNS abdominal ganglion controls ovulation in Drosophila melanogaster

Octopamine is an important neuroactive substance that modulates several physiological functions and behaviors of invertebrate species. Its biosynthesis involves two steps, one of which is catalyzed by Tyramine β-hydroxylase enzyme (TBH). The Tβh gene has been previously cloned from Drosophila melanogaster, and null mutations have been generated resulting in octopamine-less flies that show profound female sterility. This study shows that ovulation process is defective in the mutant females resulting in blockage of mature oocytes within the ovaries. The phenotype is conditionally rescued by expressing a Tβh cDNA under the control of a hsp70 promoter in adult females. Fertility of the mutant females is also restored when TBH is expressed, via the GAL4-UAS system, in cells of the CNS abdominal ganglion that express TBH and produce octopamine. This neuronal population differs from the dopamine- and serotonin-expressing cells indicating distinct patterns of expression and function of the three substances in the region. Finally, this study demonstrates that these TBH-expressing cells project to the periphery where they innervate the ovaries and the oviducts of the reproductive system. The above results point to a neuronal focus that can synthesize and release octopamine in specific sites of the female reproductive system where the amine is required to trigger ovulation (Monastirioti, 2003; Full text of article).

The effect of octopamine on ovulation could be due to its direct action on the activity of the oviductal muscles or indirectly through its putative effects on the endocrine system. In the first case, octopamine could be locally released in the reproductive system or could target it through haemolymph. The female reproductive system is extensively innervated by neurons located in the abdominal ganglion of the thoracic CNS. In contrast, several unpaired octopamine-immunoreactive (OA-IR) cells have been detected in the ventral midline of this ganglion (AC, abdominal cluster). Thus it was questioned whether any of these neurons innervate the reproductive system. Immunohistochemistry was performed in whole-mount preparations of wild type female thoracic CNS and reproductive system joined with intact abdominal nerves using the anti-TBH antibody that specifically detects OA cells in all parts of the CNS (Monastirioti, 1996) and OA processes in the larval body wall muscles (Monastirioti, 2003).

Several TBH immunoreactive (TBH-IR) cell bodies are detected in the ventral midline of the abdominal region of the adult thoracic CNS, where the OA cells have been previously detected. These cells send axons toward the dorsal neuropil, and then into the abdominal nerve. The IR axons reach the reproductive system where they ramify and target different parts of it. The immunoreactivity is primarily localized in the oviducts and ovaries. Strings of IR vesicles are observed in the ovarian sheath, on both lateral oviducts and common oviduct, while a dense network of IR fibers is primarily detected at the base of both ovaries (calyx). In contrast, no immunoreactivity was detected on the uterus muscles. TBH-IRy was evident in the reproductive system of newly emerged females (less than 8 h old), while it is missing from the respective regions in the TβhnM18 mutant females. The immunoreactivity pattern of the TBH-expressing cells and axons suggests that octopamine is being locally released in the vicinity of ovaries/oviducts where it might modulate contractility of the visceral muscles, thus influencing the ovulation process (Monastirioti, 2003).

Mechanism of the insect enzyme, tyramine beta-monooxygenase, reveals differences from the mammalian enzyme, dopamine beta-monooxygenase

Tyramine βmonooxygenase (TβM) catalyzes the synthesis of the neurotransmitter, octopamine, in insects. Kinetic and isotope effect studies have been carried out to determine the kinetic mechanism of TβM for comparison with the homologous mammalian enzymes, dopamine β-monooxygenase and peptidylglycine {alpha}-hydroxylating monooxygenase. A new and distinctive feature of TβM is very strong substrate inhibition that is dependent on the level of the co-substrate, O2, and reductant as well as substrate deuteration. This has led to a model in which tyramine can bind to either the Cu(I) or Cu(II) forms of TβM, with substrate inhibition ameliorated at very high ascorbate levels. The rate of ascorbate reduction of the E-Cu(II) form of TβM is also reduced at high tyramine, leading to the proposal of the existence of a binding site for ascorbate to this class of enzymes. These findings may be relevant to the control of octopamine production in insect cells (Hess, 2008).

Characterization of Drosophila tyramine β-hydroxylase gene and isolation of mutant flies lacking octopamine

Octopamine is likely to be an important neuroactive molecule in invertebrates. This paper reports the molecular cloning of the Drosophila melanogaster gene, which encodes tyramine β-hydroxylase (TBH), the enzyme that catalyzes the last step in octopamine biosynthesis. The deduced amino acid sequence of the encoded protein exhibits 39% identity to the evolutionarily related mammalian dopamine β-hydroxylase enzyme. A polyclonal antibody was genereated against the protein product of Tβh gene, and it was demonstrated that the TBH expression pattern is remarkably similar to the previously described octopamine immunoreactivity in Drosophila. Null mutations at the Tβh locus were created that result in complete absence of TBH protein and blockage of the octopamine biosynthesis. Tβh-null flies are octopamine-less but survive to adulthood. They are normal in external morphology, but the females are sterile, because although they mate, they retain fully developed eggs. Finally, this defect in egg laying is associated with the octopamine deficit, because females that have retained eggs initiate egg laying when transferred onto octopamine-supplemented food (Monastirioti, 1996; full text of article).


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