InteractiveFly: GeneBrief

Tyrosine decarboxylase 1 and Tyrosine decarboxylase 2: Biological Overview | References

BIOLOGICAL OVERVIEW

The trace biogenic amine tyramine is present in the nervous systems of animals ranging in complexity from nematodes to mammals. Tyramine is synthesized from tyrosine by the enzyme tyrosine decarboxylase (TDC), a member of the aromatic amino acid family, but this enzyme has not been identified in Drosophila or in higher animals. To further clarify the roles of tyramine and its metabolite octopamine, two TDC genes were cloned from Drosophila melanogaster, dTdc1 and dTdc2. Although both gene products have TDC activity in vivo, dTdc1 is expressed nonneurally, whereas dTdc2 is expressed neurally. Flies with a mutation in dTdc2 lack neural tyramine and octopamine and are female sterile due to egg retention. Although other Drosophila mutants that lack octopamine retain eggs completely within the ovaries, dTdc2 mutants release eggs into the oviducts but are unable to deposit them. This specific sterility phenotype can be partially rescued by driving the expression of dTdc2 in a dTdc2-specific pattern, whereas driving the expression of dTdc1 in the same pattern results in a complete rescue. The disparity in rescue efficiencies between the ectopically expressed Tdc genes may reflect the differential activities of these gene products. The egg retention phenotype of the dTdc2 mutant and the phenotypes associated with ectopic dTdc expression contribute to a model in which octopamine and tyramine have distinct and separable neural activities (Cole, 2005).

Tyramine, octopamine, and other 'trace' biogenic amines are found in most organisms, including bacteria, fungi, and plants, and in animals ranging in complexity from nematodes to mammals. Despite their wide distribution, the biological roles of trace amines are poorly understood. In vertebrates, trace amines are present in low levels and can displace classical biogenic amines from their stores and stimulate outward neurotransmitter efflux from biogenic amine transporters. Although there are currently no data to suggest that there are dedicated synapses for trace amines in the brain, the recent discovery of G-protein-coupled receptors that are selectively activated by trace amines, along with the presence of trace amine binding sites in the brain, indicates that they may function independently of classical neurotransmitters. A large body of clinical literature suggests a role for tyramine in the etiology of several psychiatric disorders, and the expression pattern of TA₁, a trace amine receptor potently activated by tyramine, supports such a role (Cole, 2005 and references therein).

In invertebrates, little is known about the functional role of tyramine, although distinct tyraminergic patterns of expression are thought to exist in locusts and C. elegans, and receptors with preferential response to tyramine have been identified in many insects. Additionally, tyramine affects chloride permeability in the Drosophila Malpighian tubule, relaxation of muscle tone in the locust oviduct, and behavioral responses to cocaine in Drosophila (Cole, 2005 and references therein).

Octopamine in invertebrates is often considered to be the functional homolog of vertebrate norepinephrine and is involved in a diverse range of physiological processes, including Drosophila female fertility. Flies that lack a functional octopamine receptor or cannot synthesize octopamine due to a null allele of tyramine α-hydroxylase (Tαh) show a complete egg retention phenotype. The sterility of Tαh null females is due to a defect in ovulation, which can be rescued by octopamine feeding or by driving the ectopic expression of a Tαh transgene in several driver lines that show overlapping expression in the thoracic tip of the CNS (Cole, 2005).

The first step in octopamine biosynthesis is catalyzed by tyrosine decarboxylase (TDC), but this enzyme has not been identified in Drosophila or in higher animals. Based upon sequence similarity to plant TDCs, two Drosophila melanogaster TDC genes, dTdc1 and dTdc2, have been cloned. Although both gene products have TDC activity in vivo, dTdc1 is expressed nonneurally, whereas dTdc2 is expressed neurally. Flies with a mutation in dTdc2 lack neural tyramine and octopamine and are female sterile due to egg retention; however, unlike Tαh mutants, the sterility of dTdc2 mutant females is not due to a defect in ovulation, since eggs are often found in the oviducts. Transgenic rescue of this phenotype can be accomplished by expression of either dTdc1 or dTdc2 with a dTdc2-specific driver. The specific egg retention phenotype of the dTdc2 mutant and the phenotypes associated with ectopic dTdc expression contribute to a model in which neurally derived octopamine and tyramine have distinct functions (Cole, 2005).

Therefore dTdc1 is primarily expressed in nonneural abdominal organs, whereas dTdc2 is expressed in the CNS and innervates the female reproductive tract. dTdc2^RO54, a point mutation in dTdc2, results in a loss of neural tyramine and octopamine and leads to female sterility. The ectopic expression of a dTdc1 transgene results in a complete rescue of the sterility phenotype, whereas the expression of a dTdc2 transgene leads to high levels of brain tyramine and octopamine but only partial rescue of the sterility phenotype. The difference in rescue efficiency between the genes shows that the level of expression of a TDC enzyme is as critical as the pattern of expression (Cole, 2005).

Tryptophan hydroxylase and tyrosine hydroxylase catalyze the first and rate-limiting steps in the serotonin and dopamine synthesis pathways. In the CNS, these enzymes are highly regulated, which prevents depletion of the essential amino acids tyrosine or tryptophan, respectively. Aromatic l-amino acid decarboxylase (AADCs) are responsible for the final enzymatic step in the pathways and convert 5-hydroxytryptophan to serotonin and L-DOPA to dopamine. AADCs are not rate-limiting, and although they are thought to be relatively nonselective, they are subject to short term regulatory mechanisms. TDCs are closely related to the AADC family of enzymes; however, unlike the AADCs, they act directly on tyrosine and must be highly regulated in order to maintain sufficient concentrations of neural tyrosine (Cole, 2005).

This requirement for acute regulation of TDCs complicates attempts to rescue the dTdc2^RO54 mutant, which is more readily rescued by ectopic expression of dTdc1 than by dTdc2. The simplest explanation is that the GAL4-UAS rescue paradigm more effectively controls the cellular pattern of expression than the transcriptional level of expression within particular neurons. The level of expression depends upon several factors, including the insertion site of the transgene and the number of UAS sequences within the target transgene. To control for insertion site variation, two separate insertions were used for each of the UAS lines; in each case, the two insertions give comparable results. However, because the UAS target vectors contain five tandem repeats of the GAL4 binding sequences to generate high levels of activation, dTdc1 and -2 were expressed at potentially much higher levels than they would normally be expressed. In this situation, an unregulated Tdc could lead to depletion of the essential amino acid tyrosine, which could have adverse functional consequences for the cell. It is suspected that this may be the case for the ectopic expression of dTdc2, which results in abnormally high levels of brain tyramine and octopamine but only partial functional rescue. Alternately, increased levels of tyramine in these neurons could have an inhibitory effect on egg laying, as seen in C. elegans. In fact, the abnormally high levels of neural tyramine in null tyramine β hydroxylase null Tαh^nM18 females could contribute to their complete lack of ovulation (Cole, 2005).

In contrast, the dTdc2-GAL4 driven expression of dTdc1 results in an undetectable amount of tyramine and low levels of octopamine but fully rescues the sterility phenotype. It is therefore predicted that dTdc1 is a much less active enzyme. In any case, the varying degrees of rescue achieved by the ectopic expression of dTdc1 or dTdc2 indicate that both genes express TDC activity in vivo. Additionally, these results show that precise control of this rate-limiting enzyme activity is critical for normal egg laying and may be critical for proper cellular function. Consistent with this, broader ectopic expression of dTdc2 but not dTdc1 can result in embryonic lethality (Cole, 2005).

Many enzymes that are expressed in both neural and nonneural patterns are subject to differential regulation, so the idea that dTDC1 and -2 are differentially regulated is not unanticipated. Although most of these enzymes are encoded by one gene, a recently discovered exception is mammalian tryptophan hydroxylase; TPH1 is expressed in the periphery, whereas TPH2 is expressed in the brain. In contrast, mammalian AADC and Drosophila DOPA decarboxylase and tyrosine hydroxylase use unique promoters and/or alternative splicing to direct neural and nonneural expression of distinct gene products from the same gene. Taken together, the current results, along with existing evidence, suggest that enzymes responsible for both central and peripheral biogenic amine synthesis are subject to distinct regulatory mechanisms (Cole, 2005).

Octopamine is a well established neurotransmitter, neuromodulator, and neurohormone in invertebrates. In the fly, octopamine has been implicated in complex behavioral processes and in physiological processes such as ovulation and egg laying. This study supports previous work demonstrating the importance of neural octopamine in egg laying but suggests that tyramine may have an important function in this process as well. Interestingly, dTdc2^RO54 flies retain a functional dTdc1 gene that expresses at high levels in nonneural tissues. Based on an inability to detect octopamine by HPLC in these nonneural tissues and the almost exclusively neural expression of TBH, the end product of this nonneural pathway is probably tyramine. Because this nonneural tyramine does not enter the CNS and/or ovaries, where it would be converted into octopamine, it is predicted that barriers must exist to prevent tyramine diffusion (Cole, 2005).

A previous study concluded that a small population of TBH-positive ventral ganglion neurons was necessary for ovulation (Monastirioti, 2003). This conclusion was based on driving the expression of a Tαh transgene in enhancer trap lines known to have various patterns of CNS expression but without considering potential nonneural expression. If such nonneural expression exists in these lines, the expression of a Tαh transgene could lead to the synthesis of octopamine outside of the CNS. Selective diffusion of octopamine into the CNS or ovaries could not only explain the achieved rescue but could also explain the enhanced ability of octopamine versus tyramine feeding to suppress the dTdc2^RO54 sterility phenotype (Cole, 2005).

In other insects, octopamine relaxes ovarian muscles to allow the release of mature eggs into the oviducts. In the locust Locusta migratoria, this occurs via activation of octopamine-2B receptors, which leads to a rise in cAMP. In the stable fly Stomoxys calcitrans, isolated oviducts respond to octopamine with a decrease in spontaneous contractions and an increase in myogenic and neurogenic contractions of spermathecal muscles, which moved stored sperm. The coordination of oviduct muscle tone with spermathecal contractions is necessary for successful fertilization; relaxation of oviduct contractions, presumably by octopamine, allows eggs to progress toward the uterus, where fertilization can occur. The observation that dTdc2 neurons in the ventral ganglion project directly to the posterior ovariole, and oviduct musculature supports a similar role for octopamine in Drosophila. In the ovariole, relaxation of muscle tone by octopamine could allow the release of mature eggs into the lateral oviducts, whereas relaxation of muscle tone in the lateral and common oviducts could allow the passage of eggs toward the uterus (Cole, 2005).

In many insects, octopamine also plays a role in the regulation of juvenile hormone (JH) biosynthesis. JH, a sesquiterpenoid hormone, is synthesized and released from the corpora allata of adult insects and is tightly controlled at the levels of synthesis, release, and degradation. JH is involved in every major developmental transition in the insect and is essential for many aspects of reproductive function. The involvement of octopamine in JH metabolism has been clearly demonstrated in many insects; in L. migratoria and the honeybee Apis mellifera, octopamine stimulates JH biosynthesis, whereas in the cricket Gryllus bimaculatus and the cockroach Diploptera punctata, it inhibits JH production (reviewed by Gilbert, 2000). In adult A. mellifera, very fine octopamine immunoreactive fibers with varicose terminals surround each of the gland cells in the corpora allata tissue. Furthermore, in the silkworm Bombyx mori and the flour beetle Tribolium freemani, there is in vitro evidence that secretion of octopamine increases the activity of JH esterase prior to the onset of pupation (Cole, 2005 and references therein).

In Drosophila, a direct link between octopamine and JH biosynthesis is not as well defined, but indirect evidence exists for a functional relationship. Tαh^nM18 females are reported to have a significantly increased rate of JH degradation compared with wild-type females (Gruntenko, 2000; Gruntenko, 2001), leading to speculation that JH metabolism is under the control of biogenic amines. Furthermore, under stressful conditions, levels of biogenic amines including octopamine are reported to increase, whereas JH degradation decreases. Nutritional stress leads to even more severe consequences, including a delay in oocyte maturation, degradation of early vitellogenic egg chambers, accumulation of mature oocytes, and a 24-h oviposition arrest. Until JH titers are measured directly in these situations, links between biogenic amines, stress, and JH will remain speculative. It is suggested that the loss of octopamine is the primary cause of the failure to oviposit in Tdc2^RO54 and Tαh^nM18 mutants; however, it is possible that there are consequently higher JH titers in both strains that may exacerbate the phenotype (Cole, 2005).

Little is known about the functional role of tyramine, although a recent study shows that tyramine inhibits egg laying in C. elegans¹ and has specific effects on myogenic and neurogenic contractions in locust oviduct tissue. It is tempting to speculate that tyramine may also have an inhibitory effect on Drosophila egg laying. Tdc2^RO54 flies lack both octopamine and tyramine, whereas Tαh^nM18 flies lack neural octopamine but contain ~10-fold elevated tyramine (Monastirioti, 1996). The high levels of neural tyramine in Tαh^nM18 could account for the fact that Tαh^nM18 females are deficient in ovulation but Tdc2^RO54 females are not. In further support of an inhibitory role for tyramine, the ectopic expression of dTdc2 in Tdc2^RO54 females results in abnormally high levels of brain tyramine and octopamine but only partial functional rescue. This could be a result of tyrosine depletion, as discussed previously, but it is also possible that the high levels of tyramine could lead to a partial inhibition in egg laying due to decreased ovulation (Cole, 2005).

An additional Drosophila mutant inactive, which encodes a TRPV channel, has been reported to contain low levels of tyramine and octopamine and show aberrant responses to cocaine, but this study has not found altered tyramine or octopamine levels in inactive brains. Further investigation will be required to determine whether this change in biogenic amines occurs only under selected physiological conditions (Cole, 2005).

The data indicate that there are two Tdc genes in Drosophila and A. gambiae and one in C. elegans and C. briggsae, but no potential Tdc candidate genes have been identified in the available vertebrate genomes. Based on this evidence, it seems unlikely that TDCs exist in higher vertebrates. Nevertheless, it is probable that tyramine is synthesized endogenously in the CNS due to the fact that very little tyramine crosses the blood-brain barrier from food sources. Mammalian AADC is capable of catalyzing the decarboxylation of a number of substrates in addition to L-DOPA and 5-hydroxytryptophan, although not nearly as efficiently; in addition, AADC inhibitors result in reduced tyramine production. These results suggest that the primary route of tyramine synthesis in the vertebrate brain is via decarboxylation of tyrosine by AADC. Other potential tyramine synthesis pathways have been proposed, but there are no recent studies to confirm the existence of these pathways (Cole, 2005).

Although the existence of trace amines in the brain is well known, physiological roles for these amines have been difficult to establish. The identification of genes involved in tyramine and octopamine biosynthesis in Drosophila will provide the means to develop new approaches for defining functional roles for these compounds (Cole, 2005).

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 (Tdc2^RO54). Tdc2^RO54 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 (TβH^nM18) 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 TβH^nM18 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 Tdc2^RO54 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 Tdc2^RO54 mutation, they are also evident in Tdc2^RO54 heterozygotes and in flies expressing the Tdc2-Kir2.1 transgenes. The cocaine phenotype observed in Tdc2^RO54 flies is the most extreme example of hypersensitivity observed to date. In contrast to prior expectation, Tdc2^RO54 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. TβH^nM18 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 TβH^nM18 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 TβH^nM18, would act to suppress 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 Tdc2^RO54 flies was attempted by feeding tyramine or octopamine. Feeding of these trace amines readily rescues the female sterility of Tdc2^RO54 (Cole, 2005); however, feeding proved ineffective in rescuing the hypersensitivity to cocaine or the locomotor inactivity in Tdc2^RO54, 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 Tdc2^RO54 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, TβH^nM18 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 Tdc2^RO54 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).

Octopamine in male aggression of Drosophila

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 Tβh^nM18, 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. 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 Tβh^nM18 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 Tβh^nM18 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 Tβh^nM18 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, Tβh^nM18 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 15^th to the 30^th 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 norpA^P24 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 Tβh^nM18 males. Tβh^nM18 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 Tβh^nM18 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 Tβh^nM18 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 Tβh^nM18 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 Tβh^nM18 males fought against each other. To exclude genetic background as the cause for this discrepancy (their Tβh^nM18 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 Tβh^nM18 mutant stock were more aggressive than males of the current Tβh^nM18 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 Tβh^nM18 males. (1) During jumping, distance and force production of Tβh^nM18 flies is only ~50%-60% of wild-type level. Consequently, Tβh^nM18 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 Tβh^nM18 males: While rising up on their hind legs, Tβh^nM18 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. Therefore, appropriate levels of OA might be required to motivate former losers to fight again. If Tβh^nM18 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, Tβh^nM18 males performed hardly any lunges, indicating a general loss of aggressiveness independent of former experiences (Hoyer, 2008).

Whether restoring OA in Tβh^nM18 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. Tβh^nM18 females are sterile, and fecundity can be restored by feeding octopamine (Monastirioti, 1996; Monastirioti, 2003). Moreover, feeding OA successfully rescues a memory deficit of Tβh^nM18 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 Tβh^nM18 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).

Tβh^nM18 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 Tβh^nM18 flies. Heat-shock-induced expression of Tβh in adult Tβh^nM18 males restored aggression to a small but significant extent compared to both males of the same genotype without heat shock and to heat-shocked Tβh^nM18 males lacking the hsp-Tβh construct. 47% of all mutant Tβh^nM18 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 Tβh^nM18 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 Tβh^nM18 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, 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 Tβh^nM18 males, whether the increased TA, rather than the lack of OA in Tβh^nM18 males, might have caused the aggression phenotype was examined. To address this issue, mutants of the tyrosine decarboxylase 2 (Tdc2) gene (Tdc2^RO54) were used. Tyrosine decarboxylase catalyzes the first step in octopamine biosynthesis. Tyrosine decarboxylase 2 (TDC2) converts tyrosine to TA in neurons. HPLC measurements reveal no detectable levels of TA and OA in Tdc2^RO54 mutant brains (Cole, 2005). Males homozygous for a mutation in the nearby cinnabar gene (cn¹) were used as a control because the Tdc2^RO54 mutant also carried it. Tdc2^RO54cn¹ males were strongly reduced in aggression compared to Tdc2^RO54cn¹ 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 Tdc2^RO54 and Tβh^nM18 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 Tdc2^RO54 males with TA/OA-supplemented food during adulthood again did not restore aggression. The same feeding protocol, however, rescued Tdc2^RO54 female sterility. The applied protocol has been demonstrated to restore brain TA and OA levels of Tdc2^RO54 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 Tdc2^RO54 males carrying either only the Tdc2-GAL4 transgene or the UAS-Tdc1 construct. Tdc2^RO54, Tdc2-GAL4;UAS-Tdc1 males lunged at a rate of 3% compared to the heterozygote controls, whereas Tdc2^RO54 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 Tdc2^RO54, 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 Tβh^nM18 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 Tβh^nM18 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-shi^ts1 males compared to males of the same genotype fighting at the permissive temperature of 25°C. However, using the UAS-shi^ts1 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-shi^ts1 males with Tdc2-GAL4/UAS-shi^ts1 males at the high temperature, which required a manual evaluation. The marginal decrease in aggression found for UAS-shi^ts1 males at 25°C compared to Tdc2-GAL4 males is presumably due to the slightly higher walking activity in UAS-shi^ts1 males because the pure number of lunges was not affected. Despite the problems with using the UAS-shi^ts1 transgene, the results obtained with both UAS-Kir2.1 and UAS-shi^ts1 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 Tβh^nM18 males a deficit in various aggressive behaviors when put together with control males. Tβh^nM18 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 Tβh^nM18 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 Tβh^nM18 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 Tβh^nM18 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 Tβh^nM18 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 Tdc2^RO54 males lacking both neuronal OA and TA, aggression was as much reduced as seen in Tβh^nM18 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 Tβh^nM18 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 Tβh^nM18 males via heat shock restored aggression to a small but significant number. Also expressing UAS-Tdc1 in Tdc2-neurons in Tdc2^RO54 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 15^th to the 30^th 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).

Neuroarchitecture of aminergic systems in the larval ventral ganglion of Drosophila melanogaster

Biogenic amines are important signaling molecules in the central nervous system of both vertebrates and invertebrates. In the fruit fly Drosophila melanogaster, biogenic amines take part in the regulation of various vital physiological processes such as feeding, learning/memory, locomotion, sexual behavior, and sleep/arousal. Consequently, several morphological studies have analyzed the distribution of aminergic neurons in the CNS. Previous descriptions, however, did not determine the exact spatial location of aminergic neurite arborizations within the neuropil. The release sites and pre-/postsynaptic compartments of aminergic neurons also remained largely unidentified. This study used gal4-driven marker gene expression and immunocytochemistry to map presumed serotonergic (5-HT), dopaminergic, and tyraminergic/octopaminergic neurons in the thoracic and abdominal neuromeres of the Drosophila larval ventral ganglion relying on Fasciclin2-immunoreactive tracts as three-dimensional landmarks. With tyrosine hydroxylase- (TH) or tyrosine decarboxylase 2 (TDC2)-specific gal4-drivers, the distribution of ectopically expressed neuronal compartment markers was examined in presumptive dopaminergic TH and tyraminergic/octopaminergic TDC2 neurons, respectively. The results suggest that thoracic and abdominal 5-HT and TH neurons are exclusively interneurons whereas most TDC2 neurons are efferent. 5-HT and TH neurons are ideally positioned to integrate sensory information and to modulate neuronal transmission within the ventral ganglion, while most TDC2 neurons appear to act peripherally. In contrast to 5-HT neurons, TH and TDC2 neurons each comprise morphologically different neuron subsets with separated in- and output compartments in specific neuropil regions. The three-dimensional mapping of aminergic neurons now facilitates the identification of neuronal network contacts and co-localized signaling molecules, as exemplified for DOPA decarboxylase-synthesizing neurons that co-express crustacean cardioactive peptide and myoinhibiting peptides (Vömel, 2008).

This study used gal4-driven marker gene expression and immunocytochemistry to three-dimensionally map presumed serotonergic, dopaminergic and tyraminergic/octopaminergic neurons within the Fas2 landmark system of the larval VG. Furthermore, several ectopically expressed pre- and postsynaptic markers were employed to reveal the in- and output compartments of presumptive dopaminergic TH and tyraminergic/octopaminergic TDC2 neurons. The results allow comparison of the segmental distribution patterns of aminergic neurons and to trace aminergic projections to defined neuropil areas within the VG. In the following, the morphology of aminergic neurons are related to known biogenic amine (BA) functions and describes putative neuronal network interactions with other VG neurons. This work also exemplifies how Fas2-based mapping can simplify the identification of co-localized signaling molecules, and allocate all neurons within the complex Ddc-gal4 expression pattern to distinct neuron subsets (Vömel, 2008).

Throughout the insects, similar neuron groups synthesize BAs. These groups typically comprise only few neurons with large branching patterns. In agreement with previous studies, 5-HT neurons in t1-a8 of the Drosophila larval VG represent interneurons with intrasegmental neurites. The 5-HT neurons of a8, however, appear to supply only the neuropil of a7, but not that of a8 and the adjacent 'terminal plexus'. Like 5-HT neurons, the presumptive dopaminergic TH neurons lack peripheral projections and appear to exclusively represent interneurons. In contrast, presumptive tyraminergic/octopaminergic TDC2 neurons mostly represent efferent vumTDC2 neurons. The vumTDC2 neurons obviously project to larval body wall muscles including M1 and M2 since these muscles showed TA- and OA-immunoreactive type II boutons. In a8, dorsally located dmTDC2 neurons send axons through the associated segmental nerves, and hence are efferent neurons as well. These dmTDC2 neurons probably innervate the reproductive tract in the adult female fly. Besides the dmTDC2 neurons of a8, typically two additional dmTDC2 neurons reside in the dorsal cortex between the last subesophageal neuromere and t1. These dmTDC2 neurons were not described in previous morphological studies on TA- and OA-/TβH-immunoreactive neurons. Nevertheless, all dmTDC2 neurons in the VG consistently showed strong Tdc2-gal4-driven mCD8GFP expression as well as TßH immunoreactivity. Thus, they likely synthesize both TA and OA. Although their neurites could not be traced, the dmTDC2 neurons resemble a pair of anterior medial neurons in locusts and crickets that localize to t1 and innervate the anterior connectives. Alternatively, dmTDC2 neurons may correspond to a single dorsal unpaired median neuron which resides in t1 of the locust and supplies the subesophageal nerves. Like dmTDC2 neurons, pmTDC2 neurons are probably interneurons as well. The soma position of pmTDC2 neurons highly resembles that of descending OA-immunoreactive interneurons detected in the subesophageal and thoracic neuromeres of bees, crickets, cockroaches, locusts, and moths (Vömel, 2008).

Within the larval VG of Drosophila, aminergic neurons typically show a segmentally reiterated distribution. The number of aminergic modules, however, often varies between different neuromeres. 5-HT neurons, for instance, typically occur as two bilateral pairs per neuromere. Yet, t1 comprises three 5-HT neuron pairs and a8 only one pair. The presumptive dopaminergic TH neurons also lack a strict serial homology since three ventral median TH (vmTH) neurons are present in t1, but only one in t2-a7. Furthermore, dlTH neurons locate to a1-7, but appear to be missing in t1-3. The neuromere a8 lacks TH neurons. The number of presumptive tyraminergic/octopaminergic TDC2 neurons differs between various neuromeres as well. Whereas t1 comprises one or two dmTDC2 neurons, comparable neurons are absent in t2-a7. Putative descending pmTDC2 interneurons localize to t1-a1, but appear to be missing in the remaining abdominal neuromeres. Taken together, the number of aminergic modules in t1 and a8 often deviated from that of t2-a7. This difference may-at least partially-reflect unique neuronal circuits in t1 and a8. While t1 specific physiological functions in larvae are unknown, a8 and the adjacent 'terminal plexus' are associated with the tail region, and hence contain a specific set of sensory neurons and motoneurons. The terminal neuromeres also supply several unique structures such as the spiracles or the anal pads (Vömel, 2008).

Besides the segmental differences in neuron number, the density of aminergic innervation and the amount of immunolabeling/marker gene expression varies between neuromeres as well. In particular, presumptive dopaminergic TH neurons show a striking neuromere-specific labeling pattern. Whereas a1-5 contain only few labeled TH projections, t1-3 and a6-7 comprise a comparably dense network of TH neurites. Similar to TH neurons, 5-HT neurons most densely innervate the neuropil of a7. Since a high extracellular concentration of 5-HT decreases the density of 5-HT-immunoreactive arborizations within the neuropil, a7 may represent a minor 5-HT release site. In contrast to a7, the neuropil of a8 and the adjacent 'terminal plexus' (which receive prominent peptidergic innervation) typically lack aminergic neurite arborizations. Consequently, larval aminergic neurons may play a subordinate role in tail-related physiological processes (Vömel, 2008).

To reveal putative synaptic in- and output zones of aminergic neurons, the neuronal compartment markers neuronal synaptobrevin-GFP, synaptotagmin 1-GFP, and Drosophila Down syndrome adhesion molecule [17.1]-GFP were employed. Neuronal synaptobrevin is a vesicle associated membrane protein that plays a role in the SNARE complex during vesicle transport and fusion with the plasma membrane. In accordance with this function, ectopically expressed neuronal synaptobrevin-GFP (SybGFP) accumulates at nerve terminals. SybGFP therefore served to define the presynaptic compartments of several Drosophila neurons, e.g. in the visual system. However, neuronal synaptobrevin is not restricted to small synaptic vesicles, but also locates to the membrane of large dense core vesicles, which contain BAs or neuropeptides. Consequently, in a7, SybGFP localized to putative release sites of presumptive serotonergic DDC neurons. SybGFP was also used to identify non-synaptic release sites in several peptidergic neurons. In aminergic neurons, the distribution of gal4-driven SybGFP highly resembled the corresponding mCD8GFP expression pattern. SybGFP localized in dotted patterns to aminergic neuron somata and associated neurites. It is therefore suggested that SybGFP does not exclusively label the presynaptic compartments of aminergic neurons. This fits to the assumption that ectopically expressed synaptic proteins can either localize to transport vesicles or non-synaptic compartments in peptidergic neurons. On the other hand, the ubiquitous distribution of SybGFP in aminergic neurites may suggest a widespread BA release/recycling from non-synaptic active sites. In mammals, BA release/recycling is not restricted to synapses. Vesicular monoamine transporters, which transport BAs into secretory vesicles, reside within neuron somata, axons, and dendrites. In Drosophila, the vesicular monoamine transporter DVMAT-A localizes to somata as well as neurites of several aminergic neurons both in the larval. Thus, the widespread distribution of SybGFP and DVMAT-A in aminergic neurons suggests that a considerable amount of aminergic vesicles resides at non-synaptic sites. Non-synaptic BA release/recycling might therefore play a major role for aminergic neuronal network signaling (Vömel, 2008).

Like neuronal synaptobrevin, synaptotagmins also represent integral membrane proteins of both small synaptic and large dense core vesicles. In Drosophila, the products of seven synaptotagmin genes localize to distinct neuronal compartments including the postsynaptic site. At the presynaptic site, synaptotagmin 1 does not participate in the SNARE complex, but acts as a Ca²⁺-sensor for synaptic vesicle fusion. Furthermore, synaptotagmin 1 appears to be the only crucial isoform for synaptic vesicle release. Consequently, a synaptotagmin 1-GFP fusion construct (SytGFP) was developed as a synaptic vesicle marker that specifically labels presynaptic sites. In aminergic neurons, the distribution pattern of SytGFP strikingly differed from the observed mCD8GFP and SybGFP labeling. Primary neurites of aminergic neurons always completely lacked SytGFP. Varicose neurite structures which were less evident in the mCD8GFP and SybGFP expression patterns showed strong SytGFP labeling. In agreement with the SytGFP distribution in other Drosophila neuron types, SytGFP hence appears to exclusively accumulate at the presynaptic sites of aminergic neurons. Thus, SytGFP represents a valuable marker to separate synapses from other neuronal compartments in aminergic neurons. However, since BA release is not restricted to synapses, SytGFP may not label all BA release sites of aminergic neurons. The sparse co-localization of SytGFP and SybGFP in aminergic neurites in fact suggests that aminergic vesicles-which are located distal to presynaptic sites-generally lack SytGFP. Consequently, non-synaptic BA release appears to be independent of synaptotagmin 1, but may depend on other synaptotagmin isoforms such as synaptotagmin α or β. The differing distribution of SytGFP and SybGFP also suggests that aminergic neurons contain several types of aminergic vesicles which are either associated with presynaptic or non-synaptic BA release. Alternatively, aminergic neurons may synthesize additional non-aminergic neurotransmitters like acetylcholine, GABA, or glutamate. Presumed octopaminergic efferent neurons, for instance, appear to release glutamate from type II terminals at the neuromuscular junction. In such neurons, SytGFP likely labels presynaptically located transmitter vesicles and may not reveal BA release sites (Vömel, 2008).

In contrast to SybGFP and SytGFP, ectopically expressed Drosophila Down syndrome adhesion molecule [17.1]-GFP (DscamGFP) localized to postsynaptic compartments and not to axons or presynaptic sites. Consequently, DscamGFP has served as dendrite marker in mushroom body lobe neurons. Aminergic neurons showed only weak DscamGFP labeling. DscamGFP primarily localized to neurites that lacked SytGFP labeling. Since SytGFP accumulates at presynaptic sites, DscamGFP appears to represent a valuable marker to define dendritic compartments in aminergic neurons (Vömel, 2008).

In 5-HT neurons, the distribution of ectopically expressed neuronal compartment markers was not examined since specific gal4 drivers are not available. The Ddc-gal4 driver induces marker gene expression not only in presumed serotonergic, but also in dopaminergic and additional peptidergic neurons. Consequently, neurites of different DDC neuron subsets overlap in specific neuropil areas. Presumptive serotonergic as well as dopaminergic DDC neurites, for instance, localize to the VG neuropil above the CI tracts. These conditions prevent an accurate description and interpretation of the compartment marker distribution in presumptive serotonergic DDC neurons. Thus, appropriate gal4 drivers (e.g. Dtph-gal4) are needed to further analyze 5-HT neuron morphology (Vömel, 2008).

5-HT neurons bifurcate strongly in the whole neuropil of t1-a7, and hence may influence various VG neurons including sensory, inter- as well as motoneurons. However, putative neuronal network contacts of 5-HT neurons were not examined since previous morphological studies on Drosophila 5-HT receptors did not describe the exact spatial location of the respective receptors in the larval VG (Vömel, 2008).

In TH neurons, the distribution of ectopically expressed mCD8GFP, SybGFP, SytGFP and DscamGFP differed only slightly. This might relate to the fact that the VG contains two different TH neuron groups, the vmTH and dlTH neurons, whose neurites contact each other at longitudinal projections. Consequently, pre- and postsynaptic compartments of both TH neuron groups appeared to overlap, e.g. at longitudinal projections next to the VL tracts. Since additional TH neurons located in the brain or subesophageal ganglia also innervate the VG, it was not possible to clarify which TH neuron group attributes to a particular neuronal projection. Several morphological findings, however, suggest that TH neurons possess distinct in- and output sites: Most strikingly, a1-5 contained less TH neurites labeled with mCD8GFP, SybGFP and DscamGFP, as compared to t1-3 and a6-7. In t1-a7, high amounts of SybGFP and SytGFP located to lateral longitudinal projections next to the VL tracts. These longitudinal TH neurites also contained a comparably high amount of DscamGFP, and hence likely represent synaptic in- as well as output compartments of different TH neuron groups. Besides lateral longitudinal TH projections, SybGFP and SytGFP also co-localized to the median neuropil between the DM/VM tracts. At least in a1-5, this neuropil area lacked DscamGFP, and hence probably represents a presynaptic output site of TH neurons. In a6-7, a comparably strong SybGFP and SytGFP labeling was observed in arborizations around transversal TH neurites. Whereas SybGFP mainly located to the dorsal branches of the transversal TH neurite loops, SytGFP and DscamGFP primarily labeled the ventral branches. Thus, the dorsal branches of the transversal TH neurite loops may represent non-synaptic DA release sites, while the ventral branches seem to comprise overlapping synaptic in- and output compartments of different TH neuron groups (Vömel, 2008).

Both vmTH and dorso-lateral TH (dlTH) neurons innervate distinct neuropil areas within the VG. The vmTH neurons send their primary neurites dorsally and then project through the dorsal part of the neuropil above Transversal projection (TP) 3. Since the dorsal neuropil comprises the dendritic compartments of most motoneurons, vmTH neurites are ideally located to modulate locomotor activity. This fits to the finding that DA application onto intact larval CNS-segmental preparations rapidly decreased the rhythmicity of CNS motor activity and synaptic vesicle release at the neuromuscular junction. Unlike vmTH neurons, dlTH neurons exclusively innervate the ventral part of the VG neuropil beneath TP 3. There, putative dendritic compartments of TH neurons mainly localize to lateral longitudinal and to transversal projections adjacent to the main output site of several afferent sensory neurons, e.g. tactile and proprioreceptive neurons. Thus, some TH neurons may receive synaptic input from specific sensory neurons. In contrast, TH neurons also seem to have output sites in the ventral part of the neuropil, and hence may influence the signal transmission between sensory neurons and interneurons. This fits to the finding that peptidergic apterous neurons, which appear to transmit sensory input from the VG to the brain, express DA receptors. Concomitantly, dendritic compartments of apterous neurons seem to reside adjacent to the putative DA release sites of TH neurons at the CI tracts. Besides the overlap between transversal TH neurites and sensory/interneuron projections in the ventral neuropil, TH neurons may influence several neuron groups at other locations within the VG. For instance, the putative synaptic output sites of TH neurons in the median neuropil between the DM/VM tracts overlap with presumptive input compartments of both interneurons and efferent neurons expressing peptides such as CCAP, corazonin, FMRFa, or MIP. Furthermore, the putative output sites at longitudinal TH projections next to the VL tracts lay adjacent to presumptive input compartments of e.g. efferent leucokininergic neurons (Vömel, 2008).

In the VG, most TDC2 neurons are efferent vumTDC2 neurons and showed a differential distribution of ectopically expressed SybGFP, SytGFP, and DscamGFP. The primary neurites and transversal projections of vumTDC2 neurons were labeled with DscamGFP, but lacked SytGFP. Therefore, these neurites likely represent dendritic input sites. This fits to the finding that vumTDC2 neurons possess output sites at larval body wall muscles. However, vumTDC2 neurites within the VG also contained high amounts of SybGFP, and hence may release TA/OA from non-synaptic sites. Besides vumTDC2 neurites, SybGFP strongly labeled longitudinal TDC2 neurites and associated arborizations in the dorso-lateral neuropil between TP 1 and 3. These TDC2 projections showed prominent SytGFP labeling and TßH immunoreactivity, but largely lacked DscamGFP. Thus, the dorsal part of the VG neuropil likely contains output compartments of TDC2 neurons. Since the larval brain seems to contain only tyramine- and no octopamine-immunoreactive neurons, these output sites likely derive from descending interneurons located in the subesophageal ganglia, dmTDC2 or pmTDC2 neurons. Noteworthy, the strong SybGFP and SytGFP labeling in TDC2 neurites projecting through the dorso-lateral neuropil of the VG overlapped with DscamGFP in transverse vumTDC2 neurites. Thus, descending TDC2 neurons may interact with vumTDC2 neurons (Vömel, 2008).

The VG comprises efferent vumTDC2 neurons as well as several putative TDC2 interneuron groups. Since all vumTDC2 neurons appear to have synapses at peripheral targets and dendrites in the dorsal neuropil, they show the typical motoneuron morphology. This corresponds to the finding that OA inhibited synaptic transmission at the neuromuscular junction by affecting both pre- and postsynaptic mechanisms. In addition, T?H mutant larvae, with altered levels of TA and OA, showed severe locomotion defects, which seemed to be linked to an imbalance between TA and OA signaling. Hence, vumTDC2 neurons likely regulate peripheral processes such as body wall muscle activity, whereas TDC2 interneurons centrally modulate the neuronal activity of motoneurons and interneurons involved in locomotor control. Interestingly, presumptive presynaptic compartments of descending TDC2 interneurons reside adjacent to transversal vumTDC2 dendrites. Thus, both TDC2 neuron groups may interact to modulate larval locomotor activity. Besides their function for locomotion, descending TDC2 neurons may also influence other neurons which project into the dorsal neuropil between TP 1 and 3. The putative output sites of TDC2 interneurons, for instance, lay adjacent to several peptidergic projections showing allatostatin-A, FMRFa, MIP or tachykinin immunoreactivity. However, nothing is known about TA/OA receptor distribution in the larval VG (Vömel, 2008).

During the morphological analysis of DDC neurons in the L3 larval VG, two DDC neuron groups were identified that obviously synthesize neither 5-HT nor DA. This corresponds to the previous finding that Ddc-gal4-driven marker gene expression is not restricted to presumptive serotonergic 5-HT and dopaminergic TH neurons. However, it cannot be excluded that the putative non-aminergic DDC neurons transiently synthesize BAs during other developmental stages. Ddc-gal4-driven mCD8GFP expression never revealed the dlTH neurons. This may relate to the fact that the onset of Ddc expression varies between different DDC neuron groups, and high DDC and TH levels do not temporally coincide. Taken together, these results suggest that-at least in the L3 larval VG-the Ddc-gal4 expression pattern 1) contains additional non-aminergic neurons, and 2) typically comprises most, but not all 5-HT and TH neurons. These particular characteristics of the Ddc-gal4 driver line should be carefully considered for the interpretation of studies that employed Ddc-gal4-driven expression to genetically manipulate serotonergic or dopaminergic neurons. Nevertheless, since all Ddc-gal4 expressing neurons within the VG showed at least faint DDC immunoreactivity, the Ddc-gal4 driver appears to restrict ectopical gene expression to DDC neurons. Noteworthy, the DDC neurons which lacked 5-HT and TH immunoreactivity showed corazonin and CCAP/MIP immunoreactivity respectively. In the moth Manduca sexta, these peptides play vital roles during ecdysis. At least the CCAP/MIP neurons are also necessary for the proper timing and execution of ecdysis behavior in Drosophila. Since dopaminergic DDC neurons regulate the titers of the molting hormones 20-hydroxyecdyson and juvenile hormone, both aminergic and peptidergic DDC neurons may interact to control ecdysis-related events. Recent findings indeed suggest that CCAP/MIP neurons modulate TH activity after eclosion to control the precise onset of tanning (Vömel, 2008).

Isoform- and cell-specific function of tyrosine decarboxylase in the Drosophila Malpighian tubule

The biogenic amine tyramine (TA) is a potent diuretic factor when applied to the Malpighian tubule (MT) of Drosophila, stimulating both urine production and transepithelial chloride conductance. Isolated MTs can respond not only to TA but also to its precursor, tyrosine; this observation led to the proposal that MTs are able to synthesize TA from applied tyrosine through the action of the enzyme tyrosine decarboxylase (TDC). In the current study it is shown that the non-neuronal isoform of TDC, Tdc1, is expressed in the principal cells of the MT. A mutant allele of Tdc1, Tdc1^f03311, was identified that reduced expression of the mature Tdc1 transcript by greater than 100-fold. MTs isolated from Tdc1^f03311 homozygous flies showed no significant depolarization of their transepithelial potential (TEP) or diuresis in response to tyrosine while retaining normal sensitivity to TA. By contrast, a previously identified null mutant allele of the neuronal TDC isoform Tdc2 had no effect on either tyrosine or TA sensitivity. To determine in which cell type of the MT Tdc1 expression is required, flies were generated carrying a UAS-Tdc1 transgene and cell-type-specific Gal4 drivers on a Tdc1^f03311 homozygous background. Rescue of Tdc1 expression in principal cells fully restored sensitivity to tyrosine whereas expression of Tdc1 in stellate cells had no rescuing effect. It is concluded that synthesis of TA by Tdc1 in the principal cells of the MT is required for physiological responses to tyrosine. TA synthesis in the MT is the first reported physiological role for Drosophila Tdc1 (Blumenthal, 2009).

Thus, Tdc1 expression is required for normal sensitivity of the Drosophila MT to tyrosine. Tubules isolated from Tdc1^f03311 homozygotes, which contain less than 1% of the normal amount of Tdc1 mRNA, show no depolarizing or diuretic responses to 1 mmol l^–1 tyrosine. This concentration of tyrosine elicited robust responses from heterozygous tubules and is near the solubility limit for tyrosine. Disruption of Tdc1 expression had no significant effect on either the depolarizations or the diuresis caused by TA application. These data, combined with an earlier demonstration that tyrosine responses could be blocked by antagonists of the TA receptor (Blumenthal, 2003), provide strong evidence that Tdc1 is required for the synthesis of TA from applied tyrosine in the MT. This represents the first demonstration of a physiological function for the Tdc1 gene in Drosophila and for a peripherally expressed TDC isoform in any invertebrate (Blumenthal, 2009).

In contrast to the phenotype observed upon disruption of Tdc1, there was no effect of mutating the neuronal isoform Tdc2. The Tdc2^RO54 mutation has previously been shown to be a null allele (Cole, 2005), but no differences were detected between Tdc2^RO54 heterozygotes and homozygotes in responses to either tyrosine or TA. This lack of a phenotype is consistent with the low level of Tdc2 expression in the MT reported in the microarray database and the inability of the Tdc2-gal4 transgene to drive detectable reporter gene expression in the MT. There is no evidence at present, therefore, for a functional role of Tdc2 in the MT (Blumenthal, 2009).

Expression of Tdc1 in the principal cells of the tubule is required for tyrosine sensitivity. Based on the observation that immunostaining with an anti-TA antibody selectively labels the principal cells, it has been speculated that these cells were the site of TA synthesis (Blumenthal, 2003). While the current work does not identify the actual location of TA synthesis, which would require immunolocalization of TDC1 protein, it does show that expression of the Tdc1 gene in the principal cells and not the stellate cells is sufficient to allow application of tyrosine to cause the activation of TA receptors. This finding is consistent with the localization of Tdc1-gal4 driven reporter gene expression to the principal cells. In all but one respect, driving Tdc1 expression in the principal cells resulted in tubules that behaved identically to Tdc1^f03311 heterozygotes; the exception was the duration of tyrosine-mediated diuresis, which was transient in the heterozygotes but appeared to be more sustained in the rescued tubules. In this respect, the rescued tubules more closely resembled Tdc2^RO54 heterozygotes and homozygotes, which also showed a sustained tyrosine-mediated diuresis. It is possible that this difference is related to the expression level of Tdc1; that disruption of one copy of the gene in the heterozygous mutants lowers the level of TDC1 protein below that required for a sustained diuresis and Gal4-mediated overexpression of the gene reverses this deficit (Blumenthal, 2009).

Does tyrosine have any effect on the MT beyond being a substrate for TDC? In insects, tyrosine can also serve as a substrate for the synthesis of tyrosine glucosides, one of which has been identified as a humoral factor in the silkmoth Bombyx mori. In addition, tyrosine can serve as a substrate for amino acid transporters, including some members of the iNAT family of electrogenic transporters; several transporters in this gene family are known to be expressed at high levels in the Drosophila MT. In the current work, however, tyrosine application caused no significant depolarization or diuresis in Tdc1 mutant tubules; thus, it appears that the only route through which applied tyrosine can cause diuresis or rapid changes in the TEP is through the production of TA (Blumenthal, 2009 and references therein).

Because Drosophila hemolymph contains a significant concentration of tyrosine, it is likely that MTs in vivo are tonically stimulated by endogenously produced TA. The current work has shown that Tdc1 expression is required for the tubule to respond to tyrosine; therefore, regulation of Tdc1 expression and the activity of the encoded enzyme are potential mechanisms by which urine secretion could be modulated in the intact fly. At present, nothing is known about the factors that regulate either levels of Tdc1 transcript or the enzymatic activity of its protein product, but such questions would now be interesting to study in the context of excretory function and osmoregulation (Blumenthal, 2009).

Search PubMed for articles about Drosophila Tyrosine decarboxylase

Alkema, M. J., Hunter-Ensor, M., Ringstad, N. and Horvitz, H. R. (2005). Tyramine functions independently of octopamine in the Caenorhabditis elegans nervous system. Neuron 46: 247-260. PubMed ID: 15848803

Baier, A., Wittek, B. and Brembs, B. (2002). Drosophila as a new model organism for the neurobiology of aggression?. J. Exp. Biol. 205: 1233-1240. PubMed ID: 11948200

Baines, R. A., Uhler, J. P., Thompson, A., Sweeney, S. T. and Bate, M. (2001). Altered electrical properties in Drosophila neurons developing without synaptic transmission. J. Neurosci. 21: 1523-1531. PubMed ID: 11222642

Blumenthal, E. M. (2003). Regulation of chloride permeability by endogenously produced tyramine in the Drosophila Malpighian tubule. Am. J. Physiol. Cell. Physiol. 284: C718-C728. PubMed ID: 12444020

Blumenthal, E. M. (2009). Isoform- and cell-specific function of tyrosine decarboxylase in the Drosophila Malpighian tubule. J. Exp. Biol. 212(Pt 23): 3802-9. PubMed ID: 19915121

Campbell, J. L. and Nash, H. A. (2001). Volatile general anesthetics reveal a neurobiological role for the white and brown genes of Drosophila melanogaster. J. Neurobiol. 49: 339-349. PubMed ID: 11745669

Certel, S. J., Savella, M. G., Schlegel, D. C. and Kravitz, E. A. (2007). Modulation of Drosophila male behavioral choice. Proc. Natl. Acad. Sci. 104: 4706-4711. PubMed ID: 17360588

Cole, S. H., Carney, G. E., McClung, C. A., Willard, S. S., Taylor, B. J. and Hirsh, J. (2005). Two functional but noncomplementing Drosophila tyrosine decarboxylase genes: distinct roles for neural tyramine and octopamine in female fertility. J. Biol. Chem. 280(15): 14948-55. PubMed ID: 15691831

Gilbert, L. I., Granger, N. A. and Roe, R. M. (2000). The juvenile hormones: historical facts and speculations on future research directions. Insect Biochem. Mol. Biol. 30: 617-644. PubMed ID: 10876106

Gruntenko, N. E., et al. (2000). Stress-reactivity and juvenile hormone degradation in Drosophila melanogaster strains having stress-related mutations. Insect Biochem. Mol. Biol. 30: 775-783. PubMed ID: 10876121

Gruntenko, N. E., Andreenkova, E. V., Monastirioti, M., and Rauschenbach, I. (2001). Biogenic amines downregulate the activity of enzymes participating in their synthesis in Drosophila adults. Dokl. Biol. Sci. 379: 382-384. PubMed ID: 12918381

Han, K. A., Millar, N. S. and Davis, R. L. (1998). A novel octopamine receptor with preferential expression in Drosophila mushroom bodies. J. Neurosci. 18: 3650-3658. PubMed ID: 9570796

Hardie, S. L., Zhang, J. X., and Hirsh, J. (2007). Trace amines differentially regulate adult locomotor activity, cocaine sensitivity, and female fertility in Drosophila melanogaster. Dev. Neurobiol. 67: 1396-1405. PubMed ID: 17638385

Hoyer, S. C., Eckart, A., Herrel, A., Zars, T., Fischer, S. A., Hardie, S. L. and Heisenberg, M. (2008). Octopamine in male aggression of Drosophila. Curr. Biol. 18(3): 159-67. PubMed ID: 18249112

Lee, H. G., Seong, C. S., Kim, Y. C., Davis, R. L. and Han, K. A. (2003). Octopamine receptor OAMB is required for ovulation in Drosophila melanogaster. Dev. Biol. 264: 179-190. PubMed ID: 14623240

Middleton, C. A., et al. (2006). Neuromuscular organization and aminergic modulation of contractions in the Drosophila ovary. BMC Biol 4: 17. PubMed ID: 16768790

Monastirioti, M., Linn, C. E. and White, K. (1996). Characterization of Drosophila tyramine beta-hydroxylase gene and isolation of mutant flies lacking octopamine. J. Neurosci. 16: 3900-3911. PubMed ID: 8656284

Monastirioti, M. (2003). Distinct octopamine cell population residing in the CNS abdominal ganglion controls ovulation in Drosophila melanogaster. Dev. Biol. 264: 38-49. PubMed ID: 14623230

Porzgen, P., Park, S. K., Hirsh, J., Sonders, M. S. and Amara, S. G. (2001). The antidepressant-sensitive dopamine transporterin Drosophila melanogaster: A Primordial carrier for catecholamines. Mol. Pharmacol. 59: 83-95. PubMed ID: 11125028

Rodriguez-Valentin, R., et al. (2006). Oviduct contraction in Drosophila is modulated by a neural network that is both, octopaminergic and glutamatergic. J. Cell Physiol. 209: 183-198. PubMed ID: 16826564

Roeder, T. (2005). Tyramine and octopamine: Ruling behavior and metabolism. Annu. Rev. Entomol. 50: 447-477. PubMed ID: 15355245

Schwaerzel, M., et al. (2003). Dopamine and octopamine differentiate between aversive and appetitive olfactory memories in Drosophila. J. Neurosci. 23(33): 10495-502. PubMed ID: 14627633

Sotnikova, T. D., et al. (2004). Dopamine transporter-dependent and -independent actions of trace amine b-phenylethylamine. J. Neurochem. 91: 362-373. PubMed ID: 15447669

Sotnikova, T. D., et al. (2005). Dopamine-independent locomotor actions of amphetamines in a novel acute mouse model of Parkinson disease. PLoS Biol. 3: e271. PubMed ID: 16050778

Stevenson, P.A., Dyakonova, V., Rillich, J., and Schildberger, K. (2005). Octopamine and experience-dependent modulation of aggression in crickets. J. Neurosci. 25: 1431-1441. PubMed ID: 15703397

Vömel, M. and Wegener, C. (2008). Neuroarchitecture of aminergic systems in the larval ventral ganglion of Drosophila melanogaster. PLoS ONE 3(3): e1848. PubMed ID: 18365004

Yellman, C., Tao, H., He, B. and Hirsh, J. (1997). Conserved and sexually dimorphic behavioral responses to biogenic amines in decapitated Drosophila. Proc. Natl. Acad. Sci. 94(8): 4131-6. PubMed ID: 9108117

date revised: 10 August 2010

Home page: The Interactive Fly © 2009 Thomas Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.