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

Abstract

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.

Figure 1

In vivo detection of exogenously applied 1.0 mM dopamine in the adult Drosophila brain: (A) applied potential vs time gives a visual representation of successive voltammograms with current viewed in false color; (B) dopamine concentration plotted over time. Dopamine concentration was determined from the measured current using an in vitro calibration average of three electrodes. The black arrow corresponds to a 1.0 s dopamine application beginning at 5.0 s.

Figure 2

Effect of 1.0 mM cocaine treatment on uptake of an exogenously applied 1.0 mM dopamine solution: (A) Representative concentration trace of exogenously applied dopamine in the wild-type fly before (baseline 1, 2) and after 1.0 mM cocaine treatment. A significant increase in dopamine concentration was observed. (B) Representative concentration trace of exogenously applied dopamine in the fmn mutant fly before (baseline 1, 2) and after cocaine treatment. No significant change was observed for the fmn mutant fly. Dopamine concentration was determined by converting the measured current using in vitro electrode calibration. The black arrow corresponds to a 1.0 s dopamine application beginning at 5.0 s. (C) Background-subtracted fast-scan cyclic voltammogram of baseline dopamine (dashed red line) compared with dopamine after 15 min of 1.0 mM cocaine treatment (solid black line) in the wild-type fly (average of 10 scans).

Figure 3

Comparison of wild-type and fmn mutant flies when 1.0 mM dopamine was exogenously applied before and after 1.0 mM cocaine treatment. There is a significant increase in normalized [DA]_max for wild-type flies vs fmn flies with cocaine treatment (mean ± SEM; two-way ANOVA; p = 0.0002 for interaction, n = 6). The black arrow corresponds to the beginning of the 1.0 mM cocaine treatment.

Figure 4

Comparison of wild-type and fmn mutant flies when 1.0 mM dopamine was exogenously applied before (baseline) and after 10 min of one of the following treatments: AHL saline only or 0.05, 0.5, or 1.0 mM cocaine solution (mean ± SEM; two-way ANOVA; p < 0.0001 for genotype, concentration, and interaction, n = 6). The bath solutions for the baseline and AHL saline treatment were identical. The AHL saline treatment was a control to ensure the [DA]_max response did not increase from a temporal effect owing to the control solution. There is a significant increase in normalized [DA]_max for wild-type flies after cocaine treatments compared with AHL saline (no cocaine) treatment (one-way ANOVA; p < 0.0001, post hoc Tukey pairwise comparisons; p < 0.0001 (∗∗∗) for the 1.0 mM cocaine treatment, n = 6; SEM for the baseline bars are too small to see). No significant change was observed in the fmn mutant flies between AHL saline (no cocaine) treatment and the three cocaine treatments (one-way ANOVA; p = 0.9, n = 6).

Figure 5

Comparison of uptake in adult Drosophila wild-type (solid) vs fmn mutant (striped) flies when 1.0 mM dopamine was exogenously applied before (baseline 1, 2) and after 1.0 mM stimulant treatment: (A) Following amphetamine treatment, the increases in normalized [DA]_max are significantly higher in wild-type flies compared with fmn mutant flies (mean ± SEM; two-way ANOVA; p = 0.005 for genotype, n = 5). Additionally, the 30 min treatment is significantly different from baseline 2 for the wild-type flies (one-way ANOVA; p = 0.03, post hoc Tukey pairwise comparisons; p < 0.05). (B) The increases in normalized [DA]_max are significantly higher in wild-type vs fmn flies following methamphetamine treatment (mean ± SEM; two-way ANOVA; p = 0.01 for genotype, n = 5−6). (C) Following methylphenidate treatment, the increases in normalized [DA]_max for wild-type compared with fmn flies are significantly higher (mean ± SEM; two-way ANOVA; p = 0.03 for interaction; p < 0.0001 for genotype, n = 5).