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). We previously reported the identification of two Tdc genes in Drosophila: the peripherally-expressed Tdc1 and the neurally-expressed Tdc2. To further clarify the neural functions of the trace amines in Drosophila, we examined normal and cocaine-induced locomotor activity 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 beta-hydroxylase gene (TbetaH(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 TbetaH(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.     

Two functional but noncomplementing Drosophila tyrosine decarboxylase genes: distinct roles for neural tyramine and octopamine in female fertility

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, we have cloned two TDC genes 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.     

Tyramine and octopamine have opposite effects on the locomotion of Drosophila larvae

Biogenic amines are believed to play important roles in producing behaviors. Although some biogenic amines have been extensively studied in both vertebrates and invertebrates, little is known about the effects of trace amines like tyramine and octopamine. We investigated how trace amines affect behaviors using quantitative morphometric methods on Drosophila Tbetah(nM18) and iav(N) mutants that have altered levels of tyramine and octopamine. Locomotion of wild-type and mutant third instar larvae was analyzed using Dynamic Image Analysis System (DIAS) software. We found that Tbetah(nM18) mutants, with elevated tyramine levels and reduced octopamine levels, had a severe locomotion phenotype. Mutant larvae spent much more time in pausing episodes than wild-type larvae and displayed a reduction in speed and linear translocation. The locomotion phenotype was partially rescued by feeding Tbetah(nM18) larvae octopamine, an effect that could be nullified with simultaneous feeding of tyramine. Feeding Tbetah(nM18) larvae yohimbine, an agent that inhibits the activity of Drosophila tyramine receptors, also improved some locomotion parameters. Feeding both octopamine and yohimbine further improved rescue efficiency. Simultaneously reducing the octopamine and tyramine levels as in iav(N) larvae, in contrast, led to a less severe behavioral phenotype than that of Tbetah(nM18) mutants. Feeding iav(N) larvae either tyramine or octopamine exerted only a minor improvement in locomotion. These results suggest that tyramine and octopamine have opposite effects on Drosophila larval locomotion regulation and that a balance between the two is important in producing normal behavior.     

Influence of the biogenic amine tyramine on ethanol-induced behaviors in Drosophila

The biogenic amine tyramine has been implicated in drug-induced behavior. The Drosophila inactive mutant is characterized by reduced tyramine and octopamine levels and is defective in cocaine sensitization. To test whether there is an overlap in the use of the amine neurotransmitter system in ethanol- and cocaine-induced behaviors, mutant analyses were extended to the phenotypic characterization of inactive and other mutants effecting the tyramine and octopamine neurotransmitter system. The inactive mutant displays increased ethanol sensitivity and is impaired in the initial startle response upon ethanol application. Furthermore, this mutant fails to regulate its alcohol-induced hyperactivity properly. In contrast to the defects seen after cocaine application, inactive mutants develop normal ethanol tolerance and sensitize to the locomotor activating effect of ethanol. The tyramine-beta-hydroxylase mutant (TbetaH) with increased tyramine and depleted octopamine levels displays normal ethanol sensitivity, a startle repression, and hyperactivates more in response to ethanol. In addition, TbetaH mutants fail to develop a tolerance to the hyperactivating effect of ethanol. Ethanol-induced sensitization does not seem to be impaired in either mutant, suggesting that tyramine is not required for this process. The comparative analysis of the phenotypes associated with inactive and TbetaH mutants suggests that the fine tuning of ethanol-induced hyperactivity can be correlated with different tyramine levels. Defects in other aspects of ethanol-induced behaviors might be due to different molecules or mechanisms.     

Influence of the biogenic amine tyramine on ethanol‐induced behaviors in Drosophila

The biogenic amine tyramine has been implicated in drug‐induced behavior. The Drosophila inactive mutant is characterized by reduced tyramine and octopamine levels and is defective in cocaine sensitization. To test whether there is an overlap in the use of the amine neurotransmitter system in ethanol‐ and cocaine‐induced behaviors, mutant analyses were extended to the phenotypic characterization of inactive and other mutants effecting the tyramine and octopamine neurotransmitter system. The inactive mutant displays increased ethanol sensitivity and is impaired in the initial startle response upon ethanol application. Furthermore, this mutant fails to regulate its alcohol‐induced hyperactivity properly. In contrast to the defects seen after cocaine application, inactive mutants develop normal ethanol tolerance and sensitize to the locomotor activating effect of ethanol. The tyramine‐β‐hydroxylase mutant (TβH) with increased tyramine and depleted octopamine levels displays normal ethanol sensitivity, a startle repression, and hyperactivates more in response to ethanol. In addition, TβH mutants fail to develop a tolerance to the hyperactivating effect of ethanol. Ethanol‐induced sensitization does not seem to be impaired in either mutant, suggesting that tyramine is not required for this process. The comparative analysis of the phenotypes associated with inactive and TβH mutants suggests that the fine tuning of ethanol‐induced hyperactivity can be correlated with different tyramine levels. Defects in other aspects of ethanol‐induced behaviors might be due to different molecules or mechanisms. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

First evidence of a membrane‐bound,tyramine and β‐phenylethylamine producing,tyrosine decarboxylase in Enterococcus faecalis: A two‐dimensional electrophoresis proteomic study

The soluble and membrane proteome of a tyramine producing Enterococcus faecalis, isolated from an Italian goat cheese, was investigated. A detailed analysis revealed that this strain also produces small amounts of β‐phenylethylamine. Kinetics of tyramine and β‐phenylethylamine accumulation, evaluated in tyrosine plus phenylalanine‐enriched cultures (stimulated condition), suggest that the same enzyme, the tyrosine decarboxylase (TDC), catalyzes both tyrosine and phenylalanine decarboxylation: tyrosine was recognized as the first substrate and completely converted into tyramine (100% yield) while phenylalanine was decarboxylated to β‐phenylethylamine (10% yield) only when tyrosine was completely depleted. The presence of an aspecific aromatic amino acid decarboxylase is a common feature in eukaryotes, but in bacteria only indirect evidences of a phenylalanine decarboxylating TDC have been presented so far. Comparative proteomic investigations, performed by 2‐DE and MALDI‐TOF/TOF MS, on bacteria grown in conditions stimulating tyramine and β‐phenylethylamine biosynthesis and in control conditions revealed 49 differentially expressed proteins. Except for aromatic amino acid biosynthetic enzymes, no significant down‐regulation of the central metabolic pathways was observed in stimulated conditions, suggesting that tyrosine decarboxylation does not compete with the other energy‐supplying routes. The most interesting finding is a membrane‐bound TDC highly over‐expressed during amine production. This is the first evidence of a true membrane‐bound TDC, longly suspected in bacteria on the basis of the gene sequence.     

Purification and partial gene sequence of the tyrosine decarboxylase of Lactobacillus brevis IOEB 9809

Some lactic acid bacteria contain a tyrosine decarboxylase (TDC) which converts tyrosine to tyramine, a biogenic amine frequently encountered in fermented food and wine. Purification and microsequencing of the TDC of Lactobacillus brevis IOEB 9809 allowed us to determine a partial sequence of the TDC gene encoding 264 amino acids of the enzyme. Analysis of this protein sequence revealed typical features of pyridoxal phosphate-dependent amino acid decarboxylases while not any known decarboxylase was closely related to the TDC of L. brevis IOEB 9809. In addition, we could detect other L. brevis strains carrying a TDC gene in a rapid assay based on the polymerase chain reaction.     

The effect of mutations altering biogenic amine metabolism in Drosophila on viability and the response to environmental stresses

Dopamine (DA) content, tyrosine decarboxylase (TDC) activity and survival were studied under normal and environmental stress conditions in the ste and e strains carrying ebony mutation increasing DA level and the octopamineless strain Tbetah(nM18) of Drosophila melanogaster. Wild-type strains Canton S and Oregon R, and strain p845 from which Tbetah(nM18) strain was derived were used as controls. Sexual dimorphism of TDC activity, DA content, and survival in flies of all D. melanogaster strains under study was found. Tbetah(nM18) mutation sharply reduced TDC activity in females, while ebony had no such effect. DA content and survival under heat stress in Tbetah(nM18) flies did not differ from those in the wild type. ste and e flies had drastically increased DA content under normal conditions, dramatically decreased survival under heat stress, but increased survival under starvation. DA content and survival under heat stress were also studied in the reciprocal hybrids (males) F(1) of the cross D. virilis strains 101 (wild type) and 147 with X-linked mutation, which significantly increases DA content. 147x101 males had a considerably higher DA content and lower survival than 101x147 ones. Individuals of all D. melanogaster strains under study developed the stress reaction, as judged by changes in TDC activity and DA levels. The role of biogenic amines in the stress reaction development and adaptation to environmental stresses in Drosophila is discussed. Arch. Insect Biochem. Physiol. 55:55-67, 2004.     

Type II cAMP-dependent protein kinase-deficient Drosophila are viable but show developmental, circadian, and drug response phenotypes

We identified a unique type II cAMP-dependent protein kinase regulatory subunit (PKA-RII) gene in Drosophila melanogaster and a severely hypomorphic if not null mutation, pka-RII(EP(2)2162). Extracts from pka- RII(EP(2)2162) flies selectively lack RII-specific autophosphorylation activity and show significantly reduced cAMP binding activity, attributable to the loss of functional PKA-RII. pka-RII(EP(2)2162) shows 2-fold increased basal PKA activity and approximately 40% of normal cAMP-inducible PKA activity. pka-RII(EP(2)2162) is fully viable but displays abnormalities of ovarian development and multiple behavioral phenotypes including arrhythmic circadian locomotor activity, decreased sensitivity to ethanol and cocaine, and a lack of sensitization to repeated cocaine exposures. These findings implicate type II PKA activity in these processes in Drosophila and imply a common role for PKA signaling in regulating responsiveness to cocaine and alcohol.     

Ectopic G-protein expression in dopamine and serotonin neurons blocks cocaine sensitization in Drosophila melanogaster

Sensitization to repeated doses of psychostimulants is thought to be an important component underlying the addictive process in humans [1] [2] [3] [4]. In all vertebrate animal models, including humans [5], and even in fruit flies, sensitization is observed after repeated exposure to volatilized crack cocaine [6]. In vertebrates, sensitization is thought to be initiated by processes occurring in brain regions that contain dopamine cell bodies [2] [7]. Here, we show that modulated cell signaling in the Drosophila dopamine and serotonin neurons plays an essential role in cocaine sensitization. Targeted expression of either a stimulatory (Galpha(s)) or inhibitory (Galpha(i)) Galpha subunit, or tetanus toxin light chain (TNT) in dopamine and serotonin neurons of living flies blocked behavioral sensitization to repeated cocaine exposures. These flies showed alterations in their initial cocaine responsiveness that correlated with compensatory adaptations of postsynaptic receptor sensitivity. Finally, repeated drug stimulation of a nerve cord preparation that is postsynaptic to the brain amine cells failed to induce sensitization, further showing the importance of presynaptic modulation in sensitization.     

Isolation, properties and behaviour of tyramine-producing lactic acid bacteria from wine

Wines containing high levels of biogenic amines were investigated for the presence of tyramine-producing strains. Two different Lactobacillus brevis (IOEB 9809 and IOEB 9901) able to produce the amine were isolated. None of the isolated strains identified as Oenococcus oeni formed tyramine. In addition, other Lact. brevis and Lact. hilgardii strains from our collection (IOEB) and the American Type Culture Collection (ATCC) were strong tyramine producers. Lactobacillus brevis IOEB 9809 and Lact. hilgardii IOEB 9649 were found to produce tyramine and phenylethylamine simultaneously. The conditions that can influence tyramine formation in wine were evaluated for three strains of Lact. brevis (IOEB 9809 and IOEB 9901) and Lact. hilgardii (IOEB 9649). Tyrosine was the major factor affecting tyramine formation and was enhanced by the presence of sugars, mainly glucose. Tyrosine decarboxylase (TDC) activity greatly depended on the presence of the precursor, which suggested that tyrosine induced the TDC system. These results indicate that Lactobacillus could be the lactic acid bacteria responsible for tyramine production in wine.     

Enhanced phosphorylation and enzymatic activity of phosphoglucomutase by the Btk29A tyrosine kinase in Drosophila

The Drosophila Btk29A tyrosine kinase is suggested to be involved in diverse processes, although its target proteins are unknown. In the present study, we investigated substrates of Btk29A tyrosine kinase by expressing a catalytically activated form of Btk29A-P1 (Btk-EG) in Drosophila compound eyes. Expression in eye disks led to the development of the rough-eye phenotype and increased tyrosine phosphorylation of a 65-kDa protein. Partial amino acid sequence analysis of this protein showed that it was phosphoglucomutase. Phosphoglucomutase activity in heads from Btk-EG-expressing flies was higher than that in controls, suggesting that the levels of tyrosine phosphorylation and activity of the enzyme are associated with Btk29A tyrosine kinase activity.     

Wound-inducible biosynthesis of phytoalexin hydroxycinnamic acid amides of tyramine in tryptophan and tyrosine decarboxylase transgenic tobacco lines

The wound-activated biosynthesis of phytoalexin hydroxycinnamic acid amides of tyramine was compared in untransformed and transgenic tobacco (Nicotiana tabacum) lines that express tryptophan decarboxylase (TDC), tyrosine decarboxylase (TYDC), or both activities. Transgenic in vitro-grown tobacco lines expressing TDC activity accumulated high levels of tryptamine but not hydroxycinnamic amides of tryptamine. In contrast, transgenic tobacco lines expressing TYDC accumulated tyramine as well as p-coumaroyltyramine and feruloyltyramine. The MeOH-soluble and cell wall fractions showed higher concentrations of wound-inducible p-coumaroyltyramine and feruloyltyramine, especially at and around wound sites, in TYDC and TDC xTYDC tobacco lines compared to wild-type or TDC lines. All the enzymes involved in the biosynthesis of hydroxycinnamic acid amides of tyramine were found to be similarly wound inducible in all tobacco genotypes investigated. These results provide experimental evidence that, under some circumstances, TYDC activity can exert a rate-limiting control over the carbon flux allocated to the biosynthesis of hydroxycinnamic acid amides of tyramine.     

Induced tyramine overproduction in transgenic rice plants expressing a rice tyrosine decarboxylase under the control of methanol-inducible rice tryptophan decarboxylase promoter

Tyramine, one of the various biogenic amines found in plants, is derived from the aromatic L-amino acid tyrosine through the catalytic reaction of tyrosine decarboxylase (TYDC). Tyramine overproduction by constitutive expression of TYDC in rice plants leads to stunted growth, but an increased number of tillers. To regulate tyramine production in rice plants, we expressed TYDC under the control of a methanol-inducible plant tryptophan decarboxylase (TDC) promoter and generated transgenic T(2) homozygous rice plants. The transgenic rice plants showed normal growth phenotypes with slightly increased levels of tyramine in seeds relative to wild type. Upon treatment with 1% methanol, the transgenic rice leaves produced large amounts of tyramine, whereas no increase in tyramine production was observed in wild-type plants. The methanol-induced accumulation of tyramine in the transgenic rice leaves was inversely correlated with the tyrosine level. These data indicate that tyramine production in rice plants can be artificially controlled using the methanol-inducible TDC promoter, suggesting that this promoter could be used to selectively induce the expression of other proteins or metabolites in rice plants.     

A new family of insect tyramine receptors

The Drosophila Genome Project database contains a gene, CG7431, annotated to be an "unclassifiable biogenic amine receptor." We have cloned this gene and expressed it in Chinese hamster ovary cells. After testing various ligands for G protein-coupled receptors, we found that the receptor was specifically activated by tyramine (EC(50), 5x10(-7)M) and that it showed no cross-reactivity with beta-phenylethylamine, octopamine, dopa, dopamine, adrenaline, noradrenaline, tryptamine, serotonin, histamine, and a library of 20 Drosophila neuropeptides (all tested in concentrations up to 10(-5) or 10(-4)M). The receptor was also expressed in Xenopus oocytes, where it was, again, specifically activated by tyramine with an EC(50) of 3x10(-7)M. Northern blots showed that the receptor is already expressed in 8-hour-old embryos and that it continues to be expressed in all subsequent developmental stages. Adult flies express the receptor both in the head and body (thorax/abdomen) parts. In addition to the Drosophila tyramine receptor gene, CG7431, we found another closely related Drosophila gene, CG16766, that probably also codes for a tyramine receptor. Furthermore, we annotated similar tyramine-like receptor genes in the genomic databases from the malaria mosquito Anopheles gambiae and the honeybee Apis mellifera. These four tyramine or tyramine-like receptors constitute a new receptor family that is phylogenetically distinct from the previously identified insect octopamine/tyramine receptors. The Drosophila tyramine receptor is, to our knowledge, the first cloned insect G protein-coupled receptor that appears to be fully specific for tyramine.     

Tyrosine Decarboxylase and Dopa Decarboxylase in Drosophila virilis Under Normal Conditions and Heat Stress: Genetic and Physiological Aspects

The activity of tyrosine decarboxylase (TDC) and dopa decarboxylase (DDC) was studied in adults of two lines of Drosophila virilis,contrasting in their reaction to stress conditions. Differences were found in the activity of both enzymes between individuals of the examined lines. Genetic analysis of these differences was made. Each of the two enzymes was found to be controlled by a single gene or, possibly, by a block of closely linked genes. The gene responsible for TDC activity is located on one of the autosomes (excluding chromosome II). DDC activity in D. virilisis regulated by a gene located, apparently, on chromosome II. Adults of the line responding to stress by a stress reaction (r-line) were shown to react to a short-term heat stress (38°C, 60 min) by a decrease in TDC activity. TDC activity in flies of the line incapable of the stress reaction (nr-line) did not alter in such conditions. DDC activity of adults of both lines was found to be unchangeable under stress conditions.     

Characterization of rice tryptophan decarboxylases and their direct involvement in serotonin biosynthesis in transgenic rice

l-Tryptophan decarboxylase (TDC) and l-tyrosine decarboxylase (TYDC) belong to a family of aromatic l-amino acid decarboxylases and catalyze the conversion of tryptophan and tyrosine into tryptamine and tyramine, respectively. The rice genome has been shown to contain seven TDC or TYDC-like genes. Three of these genes for which cDNA clones were available were characterized to assign their functions using heterologous expression in Escherichia coli and rice (Oryza sativa cv. Dongjin). The purified products of two of the genes were expressed in E. coli and exhibited TDC activity, whereas the remaining gene could not be expressed in E. coli. The recombinant TDC protein with the greatest TDC activity showed a K _m of 0.69 mM for tryptophan, and its activity was not inhibited by phenylalanine or tyrosine, indicating a high level of substrate specificity toward tryptophan. The ectopic expression of the three cDNA clones in rice led to the abundant production of the products of the encoded enzymes, tyramine and tryptamine. The overproduction of TYDC resulted in stunted growth and a lack of seed production due to tyramine accumulation, which increased as the plant aged. In contrast, transgenic plants that produced TDC showed a normal phenotype and contained 25-fold and 11-fold higher serotonin in the leaves and seeds, respectively, than the wild-type plants. The overproduction of either tyramine or serotonin was not strongly related to the enhanced synthesis of tyramine or serotonin derivatives, such as feruloyltyramine and feruloylserotonin, which are secondary metabolites that act as phytoalexins in plants.     

Decrease in juvenile hormone level as a result of genetic ablation of the corpus allatum cells affects the synthesis and metabolism of stress related hormones in Drosophila

Previous studies have shown that juvenile hormone (JH) regulates dopamine (DA) and octopamine (OA) content in Drosophila, and we have shown the influence of an increase in JH level on DA and OA metabolism in young females of Drosophila virilis and Drosophila melanogaster. Here we investigate the effects of genetic ablation of a subset of cells in the Corpusallatum (CA, endocrine gland synthesizing JH) on the DA levels and activities of alkaline phosphatase (ALP), tyrosine hydroxylase (TH), DA-dependent arylalkylamine N-acetyltransferase (DAT) and tyrosine decarboxylase (TDC) in young D. melanogaster females under normal conditions and upon heat stress (38°С). We show that ablation of СА cells causes: (1) a decrease in ALP, TH and DAT activities, (2) an increase in DA level and (3) an increase in TDC activity in young females. The CA ablation was also found to modulate ALP, TH and TDC responses to heat stress. Mechanisms of regulation of DA and OA levels by JH in Drosophila females are discussed.     

Genetic Control of Biogenic-Amine Systems in Drosophila Under Normal and Stress Conditions

The contents of octopamine and its precursors (tyrosine and tyramine) were studied in adults of two lines of Drosophila virilis with contrasting stress responses. It was demonstrated that in individuals responding to stress by a hormonal stress reaction (line 101), the contents of octopamine and tyrosine are lower than in nonresponding flies (line 147). It was found that there is no difference between the lines in the level of tyramine under normal conditions. The dopamine response to stressor was also studied. Genetic analysis of these differences revealed that they are controlled by a single gene and that the gene is not sex-linked. The gene controlling the response was found to be linked to chromosome 6 of D. virilis.