Drosophila Dopamine Transporter Research Articles

Using in Vivo Electrochemistry To Study the Physiological Effects of Cocaine and Other Stimulants on the Drosophila melanogaster Dopamine Transporter

Dopamine neurotransmission is thought to play a critical role in addiction reinforcing mechanisms of drugs of abuse. Electrochemical techniques have been employed extensively for monitoring in vivo dopamine changes in the brains of model organisms including rats, mice, and primates. Here, we investigated the effects of several stimulants on dopamine clearance using recently developed microanalytical tools for in vivo electrochemical measurements of dopamine in the central nervous system of Drosophila melanogaster. A cylindrical carbon-fiber microelectrode was placed in the protocerebral anterior medial region of the Drosophila brain (an area dense with dopamine neurons) while a micropipette injector was positioned to exogenously apply dopamine. Background-subtracted fast-scan cyclic voltammetry was carried out to quantify changes in dopamine concentration in the adult fly brain. Clearance of exogenously applied dopamine was significantly decreased in the protocerebral anterior medial area of the wild-type fly following treatment with cocaine, amphetamine, methamphetamine, or methylphenidate. In contrast, dopamine uptake remained unchanged when identical treatments were employed in fumin mutant flies that lack functional dopamine transporters. Our in vivo results support in vitro binding affinity studies that predict these four stimulants effectively block normal Drosophila dopamine transporter function. Furthermore, we found 10 muM to be a sufficient physiological cocaine concentration to significantly alter dopamine transporter uptake in the Drosophila central nervous system. Taken together, these data indicate dopamine uptake in the Drosophila brain is decreased by psychostimulants as observed in mammals. This validates the use of Drosophila as a model system for future studies into the cellular and molecular mechanisms underlying drug addiction in humans.

A Drosophila dopamine transporter mutant, fumin (fmn), is defective in arousal regulation

Physiologically, sleep is defined by electroencephalography, and typical REM and NREM sleep occur only in mammalian and avian species. But sleep-like inactive states also appear in other vertebrates and even in invertebrates. Similar to mammalian sleep, the inactive “rest” state of insect is regulated by both circadian and homeostatic processes which were demonstrated first in the cockroach, then in the fruit fly, Drosophila melanogaster. However, this similarity is based primarily on behavioral criteria. In the course of analyzing the nature of sleep-like rest behavior in Drosophila, we discovered by serendipity hyperactive sleepless mutant flies and named them fumin (fmn), which means “sleepless” in Japanese. We then determined the nature of the mutation by forward genetics. To our surprise, we found that their behavioral phenotype is due to a genetic lesion in the dopamine transporter (DAT) gene. The elimination of DAT activity generates a continuous wakefulness in mutant flies, resulting in dramatically increased activity levels. fmn mutants are more sensitive to mechanical stimuli when they are in prolonged immobility, indicating their decreased arousal threshold. They also show attenuation in rest (sleep) rebound. Mammalian DAT is a well-known target of psychostimulants, such as amphetamine and cocaine, and is involved in wakefulness regulation. These results indicate a critical role of dopamine for the arousal state in Drosophila and highlight a similarity between insects and mammals regarding the molecular basis of arousal. The use of a Drosophila model is expected to facilitate an understanding of arousal regulations.

Cocaine affinity decreased by mutations of aromatic residue phenylalanine 105 in the transmembrane domain 2 of dopamine transporter.

Dopamine transporter (DAT) is a major target of cocaine, one of the most abused drugs. Major efforts have been focused on defining residues in DAT involved in cocaine binding. We have isolated the Drosophila melanogaster DAT (dDAT) cDNA, which is 10-fold less sensitive to cocaine than the mammalian DATs. Replacing transmembrane domain 2 (TM2) of mouse DAT (mDAT) with dDAT sequence reduced cocaine sensitivity. The reciprocal construct exhibited increased cocaine sensitivity. Switching residue 105 in TM2, a phenylalanine conserved in all mammalian DATs, to methionine, the corresponding residue in dDAT, resulted in a functional transporter with cocaine sensitivity 4-fold lower. Replacing F105 with alanine, leucine, isoleucine, serine, threonine, asparagine, or glutamine resulted in transporters with low transport activity. In contrast, changing F105 to the other aromatic residues tyrosine or tryptophan retained more than 75% transport activity and high cocaine sensitivity. Most significantly, the reciprocal construct, switching the methionine in dDAT at the corresponding residue to phenylalanine, increased cocaine sensitivity 3-fold. Finally, the mDAT mutant with a cysteine at this position had normal transport activity but exhibited cocaine sensitivity that was 15-fold lower. These results suggest that F105 in mDAT contributes to high-affinity cocaine binding. The functional cocaine-insensitive mutants provide tools for the study of the mechanism of cocaine addiction.