Neurotransmitter Transporter-Like: A Male Germline-specific SLC6 Transporter Required for Drosophila Spermiogenesis

The SLC6 class of membrane transporters, known primarily as neurotransmitter transporters, is increasingly appreciated for its roles in nutritional uptake of amino acids and other developmentally specific functions. A Drosophila SLC6 gene, Neurotransmitter transporter-like (Ntl), is expressed only in the male germline. Mobilization of a transposon inserted near the 3′ end of the Ntl coding region yields male-sterile mutants defining a single complementation group. Germline transformation with Ntl cDNAs under control of male germline-specific control elements restores Ntl/Ntl homozygotes to normal fertility, indicating that Ntl is required only in the germ cells. In mutant males, sperm morphogenesis appears normal, with elongated, individualized and coiled spermiogenic cysts accumulating at the base of the testes. However, no sperm are transferred to the seminal vesicle. The level of polyglycylation of Ntl mutant sperm tubulin appears to be significantly lower than that of wild type controls. Glycine transporters are the most closely related SLC6 transporters to Ntl, suggesting that Ntl functions as a glycine transporter in developing sperm, where augmentation of the cytosolic pool of glycine may be required for the polyglycylation of the massive amounts of tubulin in the fly's giant sperm. The male-sterile phenotype of Ntl mutants may provide a powerful genetic system for studying the function of an SLC6 transporter family in a model organism

Neurotrophic actions of dopamine on the development of a serotonergic feeding circuit in Drosophila melanogaster

<p>Abstract</p> <p>Background</p> <p>In the fruit fly, <it>Drosophila melanogaster</it>, serotonin functions both as a neurotransmitter to regulate larval feeding, and in the development of the stomatogastric feeding circuit. There is an inverse relationship between neuronal serotonin levels during late embryogenesis and the complexity of the serotonergic fibers projecting from the larval brain to the foregut, which correlate with perturbations in feeding, the functional output of the circuit. Dopamine does not modulate larval feeding, and dopaminergic fibers do not innervate the larval foregut. Since dopamine can function in central nervous system development, separate from its role as a neurotransmitter, the role of neuronal dopamine was assessed on the development, and mature function, of the 5-HT larval feeding circuit.</p> <p>Results</p> <p>Both decreased and increased neuronal dopamine levels in late embryogenesis during development of this circuit result in depressed levels of larval feeding. Perturbations in neuronal dopamine during this developmental period also result in greater branch complexity of the serotonergic fibers innervating the gut, as well as increased size and number of the serotonin-containing vesicles along the neurite length. This neurotrophic action for dopamine is modulated by the D₂dopamine receptor expressed during late embryogenesis in central 5-HT neurons. Animals carrying transgenic RNAi constructs to knock down both dopamine and serotonin synthesis in the central nervous system display normal feeding and fiber architecture. However, disparate levels of neuronal dopamine and serotonin during development of the circuit result in abnormal gut fiber architecture and feeding behavior.</p> <p>Conclusions</p> <p>These results suggest that dopamine can exert a direct trophic influence on the development of a specific neural circuit, and that dopamine and serotonin may interact with each other to generate the neural architecture necessary for normal function of the circuit.</p

Dopamine Modulates the Rest Period Length without Perturbation of Its Power Law Distribution in Drosophila melanogaster

We analyzed the effects of dopamine signaling on the temporal organization of rest and activity in Drosophila melanogaster. Locomotor behaviors were recorded using a video-monitoring system, and the amounts of movements were quantified by using an image processing program. We, first, confirmed that rest bout durations followed long-tailed (i.e., power-law) distributions, whereas activity bout durations did not with a strict method described by Clauset et al. We also studied the effects of circadian rhythm and ambient temperature on rest bouts and activity bouts. The fraction of activity significantly increased during subjective day and at high temperature, but the power-law exponent of the rest bout distribution was not affected. The reduction in rest was realized by reduction in long rest bouts. The distribution of activity bouts did not change drastically under the above mentioned conditions. We then assessed the effects of dopamine. The distribution of rest bouts became less long-tailed and the time spent in activity significantly increased after the augmentation of dopamine signaling. Administration of a dopamine biosynthesis inhibitor yielded the opposite effects. However, the distribution of activity bouts did not contribute to the changes. These results suggest that the modulation of locomotor behavior by dopamine is predominantly controlled by changing the duration of rest bouts, rather than the duration of activity bouts

The antidepressant-sensitive dopamine transporter in Drosophila melanogaster: A primordial carrier for catecholamines

Extracellular concentrations of monoamine neurotransmitters are regulated by a family of high-affinity transporters that are the molecular targets for such psychoactive drugs as cocaine, amphetamines, and therapeutic antidepressants. In Drosophila melanogaster, cocaine-induced behaviors show striking similarities to those induced in vertebrate animal models. Although a cocaine-sensitive serotonin carrier exists in flies, there has been no pharmacological or molecular evidence to support the presence of distinct carrier subtypes for other bioactive monoamines. Here we report the cloning and characterization of a cocaine-sensitive fly dopamine transporter (dDAT). In situ hybridization demonstrates that dDAT mRNA expression is restricted to dopaminergic cells in the fly nervous system. The substrate selectivity of dDAT parallels that of the mammalian DATs in that dopamine and tyramine are the preferred substrates, whereas octopamine is transported less efficiently, and serotonin not at all. In contrast, dDAT inhibitors display a rank order of potency most closely resembling that of mammalian norepinephrine transporters. Cocaine has a moderately high affinity to the cloned dDAT (IC50 = 2.6 muM). Voltage-clamp analysis of dDAT expressed in Xenopus laevis oocytes indicates that dDAT-mediated uptake is electrogenic; however, dDAT seems to lack the constitutive leak conductance that is characteristic of the mammalian catecholamine transporters. The combination of a DAT-like substrate selectivity and norepinephrine transporter-like inhibitor pharmacology within a single carrier, and results from phylogenetic analyses, suggest that dDAT represents an ancestral catecholamine transporter gene. The identification of a cocaine-sensitive target linked to dopaminergic neurotransmission in D. melanogaster will serve as a basis for further dissection of the genetic components of psychostimulant-mediated behavior.X11113sciescopu

Characterisation of the recently cloned rat noradrenaline transporter - Comparison with bovine and human transporters

The aims of this study were to characterize the recently cloned rat norepinephrine transporter (NET) in more detail and in particular to study possible species differences in its pharmacological properties compared with the human and bovine NETs. The study was carried out by measuring the uptake of [3H]norepinephrine in COS-7 cells expressing the NET after transient transfection with rat, human, or bovine NET cDNA. There were small but significant differences between the rat NET and the human or bovine NETs with respect to the affinities of sodium ions (greater for rat than for bovine) of the substrates norepinephrine, epinephrine, and 1-methyl-4-phenylpyridinium (greater for human than for rat), and of the inhibitor cocaine (greater for human and bovine than for rat), whereas the affinities of dopamine and of most inhibitors, including tricyclic antidepressants, showed no species differences. The fact that the affinities for some substrates, cocaine and sodium ions exhibited small but significant interspecies differences among the rat, human, and bovine NETs suggests that ligand recognition, the translocation process, and sodium ion dependence are influenced differentially by just a few amino acid exchanges in the primary sequences of the transporters. On the other hand, the lack of any major differences in the pharmacological properties of the rat, human, and bovine NETs in this study suggests that data obtained in previous studies on rat tissues and bovine cells can be extrapolated, in all except the most quantitative analyses, to the properties of the human NET

Comparison of the pharmacological properties of cloned rat, human, and bovine norepinephrine transporters

The aims of this study were to characterize the recently cloned rat norepinephrine transporter (NET) in more detail and in particular to study possible species differences in its pharmacological properties compared with the human and bovine NETs. The study was carried out by measuring the uptake of [H-3]norepinephrine in COS-7 cells expressing the NET after transient transfection with rat, human, or bovine NET cDNA. There were small but significant differences between the rat NET and the human or bovine NETs with respect to the affinities of sodium ions (greater for rat than for bovine) of the substrates norepinephrine, epinephrine, and 1-methyl-4-phenylpyridinium (greater for human than for rat), and of the inhibitor cocaine (greater for human and bovine than for rat), whereas the affinities of dopamine and of most inhibitors, including tricyclic antidepressants, showed no species differences. The fact that the affinities for some substrates, cocaine and sodium ions exhibited small but significant interspecies differences among the rat, human, and bovine NETs suggests that ligand recognition, the translocation process, and sodium ion dependence are influenced differentially by just a few amino acid exchanges in the primary sequences of the transporters. On the other hand, the lack of any major differences in the pharmacological properties of the rat, human, and bovine NETs in this study suggests that data obtained in previous studies on rat tissues and bovine cells can be extrapolated, in all except the most quantitative analyses, to the properties of the human NET