Distinct neural circuits reflect sex, sexual maturity, and reproductive status in response to stress in Drosophila melanogaster.

Studies in mammalian systems have shown an array of changes in transmitter signaling in diverse brain regions in response to stress, which differ depending on the age and genetic makeup of the animal, as well as the type of stress. Here, we exploit the genetic tractability of the fruit fly, Drosophila melanogaster, a comparatively simple but useful model in which to elucidate conserved components of stress response pathways. We show that structures within the mushroom bodies and central complex, two distinct anatomical regions within the Drosophila brain, modulate behavioral responses to two different environmental stressors. Modification of behavioral output after exposure to these stressors was dependent on the sex, sexual maturity, and reproductive status of the animal. These parameters also affected whether a mutant Drosophila strain carrying specific defects within the mushroom bodies and/or central complex modified its response to stress relative to wild-type flies. Our results suggest that for each population, unique subsets of neurons are recruited into the stress response circuitry and differentially affect locomotor behavior and cardiac function. These data also provide evidence for neural plasticity in the adult insect brain.

Compartmentalization of neuronal and peripheral serotonin synthesis in Drosophila melanogaster.

In Drosophila, one enzyme (Drosophila tryptophan-phenylalanine hydroxylase, DTPHu) hydroxylates both tryptophan to yield 5-hydroxytryptophan, the first step in serotonin synthesis, and phenylalanine, to generate tyrosine. Analysis of the sequenced Drosophila genome identified an additional enzyme with extensive homology to mammalian tryptophan hydroxylase (TPH), which we have termed DTRHn. We have shown that DTRHn can hydroxylate tryptophan in vitro but displays differential activity relative to DTPHu when using tryptophan as a substrate. Recent studies in mice identified the presence of two TPH genes, Tph1 and Tph2, from distinct genetic loci. Tph1 represents the non-neuronal TPH gene, and Tph2 is expressed exclusively in the brain. In this article, we show that DTRHn is neuronal in expression and function and thus represents the Drosophila homologue of Tph2. Using a DTRHn-null mutation, we show that diminished neuronal serotonin affects locomotor, olfactory and feeding behaviors, as well as heart rate. We also show that DTPHu functions in vivo as a phenylalanine hydroxylase in addition to its role as the peripheral TPH in Drosophila, and is critical for non-neuronal developmental events.

Dopamine and sensory tissue development in Drosophila melanogaster.

Dopamine is an important signaling molecule in the nervous system; it also plays a vital role in the development of diverse non-neuronal tissues in the fruit fly Drosophila melanogaster. The current study demonstrates that males depleted of dopamine as third instar larvae (via inhibition of the biosynthetic enzyme tyrosine hydroxylase) demonstrated abnormalities in courtship behavior as adults. These defects were suggestive of abnormalities in sensory perception and/or processing. Electroretinograms (ERGs) of eyes from adults depleted of dopamine for 1 day as third instar larvae revealed diminished or absent on- and off-transients. These sensory defects were rescued by the addition of L-DOPA in conjunction with tyrosine hydroxylase inhibition during the larval stage. Depletion of dopamine in the first or second larval instar was lethal, but this was not due to a general inhibition of proliferative cells. To establish that dopamine was synthesized in tissues destined to become part of the adult sensory apparatus, transgenic lines were generated containing 1 or 4 kb of 5' upstream sequences from the Drosophila tyrosine hydroxylase gene (DTH) fused to the E. coli beta-galactosidase reporter. The DTH promoters directed expression of the reporter gene in discrete and consistent patterns within the imaginal discs, in addition to the expected expression in gonadal, brain, and cuticular tissues. The beta-galactosidase expression colocalized with tyrosine hydroxylase protein. These results are consistent with a developmental requirement for dopamine in the normal physiology of adult sensory tissues.

Dopamine and senescence in Drosophila melanogaster.

Five-day-old Drosophila melanogaster males, when exposed to 2-h-old males, will perform courtship rituals; the intensity and duration of this behavior rapidly diminishes with time. The ability of the older males to habituate to the attractive signals given off by the younger males is a dopaminergic-modulated experience-dependent modification of behavior that is abolished with increasing age. Dopamine-depleted females show increased resistance to copulation; 20-day-old females demonstrated an increase in copulation avoidance compared with younger (5-15-day-old) females. These changes in dopaminergic-modulated behaviors observed during aging parallel declines in whole body levels of dopamine. Immunocytochemical analysis of adult brains using an antibody raised against Drosophila tyrosine hydroxylase to visualize catecholaminergic cell bodies revealed increased degeneration of the cell bodies with aging. These results suggest that the deficits seen in dopaminergic-modulated behaviors may arise as a consequence of degenerative changes within the aging brain.

Dopamine modulates acute responses to cocaine, nicotine and ethanol in Drosophila.

BACKGROUND: Drugs of abuse have a common property in mammals, which is their ability to facilitate the release of the neurotransmitter and neuromodulator dopamine in specific brain regions involved in reward and motivation. This increase in synaptic dopamine levels is believed to act as a positive reinforcer and to mediate some of the acute responses to drugs. The mechanisms by which dopamine regulates acute drug responses and addiction remain unknown. RESULTS: We present evidence that dopamine plays a role in the responses of Drosophila to cocaine, nicotine or ethanol. We used a startle-induced negative geotaxis assay and a locomotor tracking system to measure the effect of psychostimulants on fly behavior. Using these assays, we show that acute responses to cocaine and nicotine are blunted by pharmacologically induced reductions in dopamine levels. Cocaine and nicotine showed a high degree of synergy in their effects, which is consistent with an action through convergent pathways. In addition, we found that dopamine is involved in the acute locomotor-activating effect, but not the sedating effect, of ethanol. CONCLUSIONS: We show that in Drosophila, as in mammals, dopaminergic pathways play a role in modulating specific behavioral responses to cocaine, nicotine or ethanol. We therefore suggest that Drosophila can be used as a genetically tractable model system in which to study the mechanisms underlying behavioral responses to multiple drugs of abuse.

The effects of 5-HT on sensory, central and motor neurons driving the abdominal superficial flexor muscles in the crayfish.

Serotonin (5-HT) induces a variety of physiological and behavioral effects in crustaceans. However, the mechanisms employed by 5-HT to effect behavioral changes are not fully understood. Among the mechanisms by which these changes might occur are alterations in synaptic drive and efficacy of sensory, interneurons and motor neurons, as well as direct effects on muscles. We investigated these aspects with the use of a defined sensory-motor system, which is entirely contained within a single abdominal segment and consists of a 'cuticular sensory neurons segmental ganglia abdominal superficial flexor motor neurons-muscles' circuit. Our studies address the role of 5-HT in altering (1) the activity of motor neurons induced by sensory stimulation; (2) the inherent excitability of superficial flexor motor neurons; (3) transmitter release properties of the motor nerve terminal and (4) input resistance of the muscle. Using en passant recordings from the motor nerve, with and without sensory stimulation, and intracellular recordings from the muscle, we show that 5-HT enhances sensory drive and output from the ventral nerve cord resulting in an increase in the firing frequency of the motor neurons. Also, 5-HT increases transmitter release at the neuromuscular junction, and alters input resistance of the muscle fibers.

Dopaminergic modulation of motor neuron activity and neuromuscular function in Drosophila melanogaster.

Dopamine is found in both neuronal and non-neuronal tissues in the larval stage of the fruit fly, Drosophila melanogaster, and functions as a signaling molecule in the nervous system. Although dopaminergic neurons in the central nervous system (CNS) were previously thought solely to be interneurons, recent studies suggest that dopamine may also act as a neuromodulator in humoral pathways. We examined both application of dopamine on intact larval CNS-segmental preparations and isolated neuromuscular junctions (NMJs). Dopamine rapidly decreased the rhythmicity of the CNS motor activity. Application of dopamine on neuromuscular preparations of the segmental muscles 6 and 7 resulted in a dose-responsive decrease in the excitatory junction potentials (EJPs). With the use of focal, macro-patch synaptic current recordings the quantal evoked transmission showed a depression of vesicular release at concentrations of 10 microM. Higher concentrations (1 mM) produced a rapid decrement in evoked vesicular release. Dopamine did not alter the shape of the spontaneous synaptic currents, suggesting that dopamine does not alter the postsynaptic muscle fiber receptiveness to the glutaminergic motor nerve transmission. The effects are presynaptic in causing a reduction in the number of vesicles that are stimulated to be released due to neural activity.

Dopamine and mushroom bodies in Drosophila: experience-dependent and -independent aspects of sexual behavior.

Depletion of dopamine in Drosophila melanogaster adult males, accomplished through systemic introduction of the tyrosine hydroxylase inhibitor 3-iodo-tyrosine, severely impaired the ability of these flies to modify their courtship responses to immature males. Mature males, when first exposed to immature males, will perform courtship rituals; the intensity and duration of this behavior rapidly diminishes with time. Dopamine is also required for normal female sexual receptivity; dopamine-depleted females show increased latency to copulation. One kilobase of 5' upstream information from the Drosophila tyrosine hydroxylase (DTH) gene, when fused to the Escherichia coli beta-galactosidase reporter and transduced into the genome of Drosophila melanogaster, is capable of directing expression of the reporter gene in the mushroom bodies, which are believed to mediate learning acquisition and memory retention in flies. Ablation of mushroom bodies by treatment of newly hatched larva with hydroxyurea resulted in the inability of treated mature adult males to cease courtship when placed with untreated immature males. However, functional mushroom bodies were not required for the dopaminergic modulation of an innate behavior, female sexual receptivity. These data suggest that dopamine acts as a signaling molecule within the mushroom bodies to mediate a simple form of learning.

Dopamine modulates female sexual receptivity in Drosophila melanogaster.

Newly eclosed Drosophila melanogaster flies were systemically depleted of dopamine by feeding an inhibitor of the biosynthetic enzyme, tyrosine hydroxylase, and analyzed for abnormalities in courtship behavior. Dopamine-depleted females were significantly less receptive to males than were control females, although males were strongly attracted to treated females. The decrease in receptivity was reversed by the addition of L-DOPA (the product of the tyrosine hydroxylation reaction) to food containing the inhibitor. Male courtship behaviors were unaffected by this treatment. Female receptivity may be regulated via interactions with hormonal pathways, since depletion of dopamine levels via inhibition of tyrosine hydroxylase activity in Drosophila melanogaster adult females has established that dopamine is required for normal ovarian maturation and fecundity.

GABA transporters in Drosophila melanogaster: molecular cloning, behavior, and physiology.

Molecular cloning of GABA transporter-homologous cDNAs from a Drosophila melanogaster head-specific library was accomplished using a conserved oligomer from a highly conserved domain within the mammalian GABA transporters. Partial DNA sequencing of these cDNAs demonstrated homology with the mammalian transporters, indicating these are ancient, evolutionarily conserved molecules. Although the Drosophila cDNAs had distinct restriction enzyme patterns, they recognized the same locus in Drosophila genomic DNA, suggesting that the multiple isoforms might arise via alternative splicing. Antibodies specific for the mammalian GABA transporters GAT 1, GAT 2 and GAT 3 recognized non-overlapping and developmentally distinct patterns of expression in Drosophila neuronal tissues. Treatment of larval instars with nipecotic acid, a generalized GABA reuptake inhibitor, revealed specific, dose-dependent alterations in behavior consistent with the presence of multiple transporter molecules with differing affinities for this drug. Synaptic current recordings revealed that nipecotic acid treated larvae have an increase in latency jitter of evoked quantal release, resulting in a broader average excitatory junctional current which was manifested in a broader EJP. These results imply that alterations in the development of the CNS occur if GABAergic neurotransmission is potentiated during development. The data suggest that, as in mammals, there are multiple GABA transporters in Drosophila whose expression is differentially regulated.

Multiple roles for dopamine in Drosophila development.

Manipulation of dopamine levels by inhibition of tyrosine hydroxylase activity was accomplished in Drosophila melanogaster larval instars by feeding enzyme inhibitors for a 24-hr period. Behavioral assays performed immediately after treatment demonstrated that larval phototaxis, salt aversion, and heptanol preference were unaffected by reduced levels of dopamine. Within a few hours of treatment, the larvae ceased exploratory behavior and were unresponsive to external stimuli; these larvae eventually died. This behavior is strikingly similar to that displayed by dopamine-deficient transgenic mice. Treated larvae placed immediately onto normal food (to replenish dopamine levels) showed significant developmental delays and decreased fertility as adults. The lethality, developmental retardation, and decrease in fertility were reversed by addition of L-DOPA to inhibitor-containing food, suggesting that these effects were due solely to inhibition of tyrosine hydroxylation. Depletion of dopamine in newly eclosed females resulted in abnormally developed ovaries. These results suggest that the enzymatic function of tyrosine hydroxylase is vital and that reduced levels of dopamine result in akinesia and lethality, developmental retardation, and decreased fertility.

Drosophila tyrosine hydroxylase is encoded by the pale locus.

We have reintroduced an 8 kb genomic fragment from the Drosophila tyrosine hydroxylase (DTH) locus into the genome of mutant pale (ple) flies. ple was first recovered as a recessive embryonic lethal by Jurgens et al. (1984) and maps to the same chromosomal region as DTH (65A-E). Mutant ple alleles affect pigmentation of the cuticle (L-DOPA, the product of the reaction catalyzed by TH, is an intermediate in the cuticular sclerotization and pigmentation pathways) and catecholamine biosynthesis. In this report we demonstrate that ple does encode the structural gene for TH, since the reintroduced sequences rescue ple flies from lethality to viable adults. Morphological, immunocytochemical, and behavioral characterization of three transformant lines suggests that the reintroduced sequences contain the necessary elements for correct temporal and spatial expression of the gene, but may not contain all the sequences essential for quantitative expression.

A single locus encodes both phenylalanine hydroxylase and tryptophan hydroxylase activities in Drosophila.

We have used a full-length clone encoding rabbit tryptophan hydroxylase (TRH) to isolate the Drosophila homologue (DTPH). Southern analysis of Drosophila genomic DNA reveals a pattern indicative of a single gene. The single transcript is expressed in adult head and body mRNA but is also detected in mRNA from early embryos. The embryonic transcript is ubiquitously expressed and appears to concentrate in yolk granules. In situ hybridization of TRH-homologous antisense RNA probe to sectioned tissue from third instar larvae demonstrated the presence of this transcript in fat body and cuticular tissue. Developmental immunoblot analysis using antibodies raised against a beta-galactosidase-Drosophila fusion protein revealed a 45-kDa embryonic protein also detected in female abdomens and a 50-kDa protein found in larval and adult stages. Immunocytochemical analysis of the Drosophila protein in the larval central nervous system showed that it appeared to be present in both serotonin- and catecholamine-containing neurons. A nonfusion protein generated in Escherichia coli hydroxylates both tryptophan and phenylalanine. We propose that there are only two aromatic amino acid hydroxylase genes in Drosophila: one encoding tyrosine hydroxylase, DTH, and DTPH, a gene encoding both tryptophan and phenylalanine hydroxylase activities.

Isolation and characterization of the gene for Drosophila tyrosine hydroxylase.

A Drosophila cDNA homologous to a rat tyrosine hydroxylase (TH) probe has been isolated and sequenced. The deduced amino acid sequence predicts a 57,861 dalton protein with almost 50% identity with rat TH. In vitro transcription of the cDNA followed by in vitro translation yields a single protein species of approximately 58,000 daltons. The in vitro translation product, as well as a protein of the same molecular weight from wild-type Drosophila head protein extracts, is recognized by an antibody made against bovine TH. The presence of TH enzymatic activity in heads was demonstrated. In situ hybridization to polytene chromosomes localized the gene to 65B. The comapping of the mutant pale to this same region, as well as its phenotype, suggests that pale may be a TH mutation.

Genetic determinants of neoplastic diseases induced by a subgroup F avian leukosis virus.

Two subgroup F avian leukosis viruses, ring-necked pheasant virus (RPV) and RAV-61, were previously shown to induce a high incidence of a fatal proliferative disorder in the lungs of infected chickens. These lung lesions, termed angiosarcomas, appear rapidly (4 to 5 weeks after infection), show no evidence of proto-oncogene activation by proviral integration, and are not induced by avian leukosis viruses belonging to other subgroups. To identify the viral sequences responsible for induction of these tumors, we constructed recombinant viruses by exchanging genomic segments of molecularly cloned RPV with those of a subgroup A leukosis virus, UR2AV. The ability to induce rapid lung tumors segregated only with the env sequences of RPV; the long terminal repeat of RPV was not required. However, recombinants carrying both env and long terminal repeat sequences of RPV induced lung tumors with a shorter latency. In several cases, recombinant viruses exhibited pathogenic properties differing from those of either parental virus. Recombinants carrying the gag-pol region of RPV and the env gene of UR2AV induced a high incidence of a muscle lesion termed infiltrative intramuscular fibromatosis. One recombinant, EU-8, which carries the gag-pol and LTR sequences of RPV, and the env gene of UR2AV, induced lymphoid leukosis after an unusually short latent period. The median time of death from lymphoid leukosis was 6 to 7 weeks after infection with EU-8 compared with approximately 5 months for UR2AV.

Proto-oncogene c-ros codes for a molecule with structural features common to those of growth factor receptors and displays tissue specific and developmentally regulated expression.

A recombinant DNA clone containing cellular sequences homologous to the transforming sequence, v-ros, of avian sarcoma virus UR2 was isolated from a chicken genomic DNA library. Heteroduplex mapping and nucleotide sequencing reveal that the v-ros sequences are distributed in nine exons ranging from 65 to 204 nucleotides on cellular ros (c-ros) DNA over a range of 11 kilobases. Comparison of the deduced amino acid sequences of c-ros and v-ros shows two differences: v-ros contains a three-amino-acid insertion within the hydrophobic domain presumed to be involved in membrane association, and (ii) the carboxyl 12 amino acids of v-ros are completely different from those of the deduced c-ros sequence. The deduced amino acid sequence of c-ros bears striking structural features similar to those of insulin and epidermal growth factor receptors, including the presumed hydrophobic membrane binding domain, amino acids flanking the domain, and the distance between the domain and the catalytic region of the kinase activity. The expression of c-ros appears to be under a very stringent control. When tissues at various stages of chicken development were analyzed, only kidney was found to contain a significant level of c-ros RNA. The level of c-ros RNA in kidney tissue is most abundant in 7- to 14-day-old chickens. Finally, nucleotide sequences of c-ros DNA and UR2-associated helper viral genome at regions corresponding to the gag ros recombination site suggest that the junction has been formed by RNA splicing.

Nucleotide sequence of avian sarcoma virus UR2 and comparison of its transforming gene with other members of the tyrosine protein kinase oncogene family.

The genome of avian sarcoma virus UR2 was completely sequenced and found to have a size of 3,165 nucleotides. The UR2-specific transforming sequence, ros, with a length of 1,273 nucleotides, is inserted between the truncated gag gene coding for p19 and the env gene coding for gp37 of the UR2AV helper virus. The deduced amino acid sequence for the UR2 transforming protein P68 gives a molecular weight of 61,113 and shows that it is closely related to the oncogene family coding for tyrosine protein kinases. P68 contains two distinctive hydrophobic regions that are absent in other tyrosine kinases, and it has unique amino acid changes and insertions within the conserved domain of the kinases. These characteristics may modulate the activity and target specificity of P68.

Molecular cloning and characterization of avian sarcoma virus UR2 and comparison of its transforming sequence with those of other avian sarcoma viruses.

Avian sarcoma virus UR2 and its associated helper virus, UR2AV , were molecularly cloned into lambda gtWES X lambda B by using unintegrated viral DNAs. One UR2 and several UR2AV clones were obtained. The UR2 DNA was subsequently cloned into pBR322. Both UR2 and UR2AV DNAs were tested for their biological activity by transfection onto chicken embryo fibroblasts. When cotransfected with UR2AV DNA, UR2 DNA was able to induce transformation of chicken embryo fibroblasts with a morphology similar to that of parental UR2 . UR2 -specific protein with kinase activity and UR2 -specific RNA were detected in the transfected cells. Transforming virus, UR2 ( UR2AV ), was produced from the doubly transfected cells. Five of the six UR2AV clones tested were also shown to be biologically active. The insert of the UR2 DNA clone is 3.4 kilobases in length and contains two copies of the long terminal repeat. Detailed restriction mapping showed that UR2 DNA shared with UR2AV DNA 0.8 kilobases of 5' sequence, including a portion of 5' gag, and 1.4 kilobases of 3' sequence, including a portion of 3' env. The UR2 transforming sequence, ros, is ca. 1.2 kilobases. No significant homology was found between v-ros and the conserved regions of v-src, v-yes, or v- abl . By contrast, a significant homology was found between v-ros and v-fps. The v-fps-related sequence was mapped within a 300-base-pair sequence in the middle of ros.

Cloning and characterization of nine heat-shock-induced mRNAs of Drosophila melanogaster.

We have cloned cDNA segments representing nine different genes whose expression is activated by heat-shock treatment of Drosophila melanogaster cells. These nine were selected from a larger population based on the fact that the levels of the polysomal, poly(A)-containing RNA homologous to each of these cDNA segments is significantly greater in induced cells. Among these genes are four that hybridize in situ to major heat-shock puff sites on polytene chromosomes, and five previously uncharacterized genes that hybridize to other sites. Some of these additional sites have been previously implicated as responders to heat shock. Hybridization of in vivo pulse-labeled. total RNA from induced and uninduced cells to specific cDNA segments provided an estimate of both the relative level of different heat-shock transcripts and the inducibility of the nine genes. Although the most abundant RNA species are derived from genes at major heat-shock puff sites, all nine show significant induction. These results indicate that the repertoire of gene expression that is activated or amplified by heat shock is not confined to these major puff sites and that the number of genes involved in the heat-shock response is much larger than the seven commonly noted.