Dissociation of rewarding and dopamine transporter-mediated properties of amphetamine

Abstract

The interaction of amphetamine (AMPH) with the dopamine (DA) transporter (DAT) is thought to be critically important for the DA-elevating actions of this drug. It is commonly believed that DA elevations are involved in the rewarding/reinforcing properties of AMPH and other drugs of abuse. Here, we found that DAT deletion did not eliminate the rewarding effects of AMPH as measured by conditioned place preference (CPP). In fact, mice in which the DAT gene has been deleted (DAT-KO mice) exhibited AMPH-induced CPP for many weeks after the time when extinction occurred in WT mice. Moreover, systemic AMPH still increased extracellular DA in the nucleus accumbens (NAc) of mice lacking the DAT, although local infusion of AMPH into the NAc did not have this effect. By using voltammetry in NAc slices, we found that AMPH did not decrease the rate of DA clearance. The rate of ventral tegmental area DA neuron firing was dramatically inhibited by AMPH in brain slices from WT mice, but there was no inhibition of firing in DAT-KO mice. AMPH-induced CPP was abolished by pretreatment with WAY-100635, a serotonin 5-HT(1A) receptor antagonist, in DAT-KO mice, but the drug did not change AMPH place preference in WT mice. Therefore, despite the absence of the DAT, AMPH displays rewarding effects and causes an increase in extracellular DA in the NAc of DAT-KO mice, acting indirectly in this case. The 5-HT system may be involved in the rewarding effects of AMPH in these mice.

Fig. 1.

Effect of AMPH at 5 mg/kg, i.p. (A), and 50 and 100 μM local infusions (B) on extracellular DA levels in the NAc of DAT-KO and WT mice, measured by microdialysis. The data are presented as mean ± SEM of the percent change from baseline (100%) from a mean of three samples from each mouse immediately before AMPH or saline (SAL) administration (n = 4 - 6 per group). NAc DA levels were significantly elevated by systemic AMPH in both WT and DAT-KO mice (P < 0.001); however, local infusion only elevated DA levels in WT mice (P < 0.001).

Fig. 2.

AMPH slows DA clearance in NAc core of WT (A) but not DAT-KO (B) mice. Electrically stimulated (single pulse) DA efflux was measured by FSCV in NAc core slices before (Lower Left) and during (Lower Right) AMPH (10 μM) bath application. (Upper) Background-subtracted cyclic voltammograms taken at the peak response. There is an oxidation peak at 600 mV and a reduction peak at -200 mV versus Ag/AgCl, identifying the released species as DA.

Fig. 3.

Effect of AMPH on DA clearance in the core and shell NAc in WT and DAT-KO mice measured by FSCV in brain slices. AMPH was applied to the NAc at 1 (gray bars) and 10 (white bars) μM. Each concentration was tested in one slice, which served as its own precondition control (black bars). For each experimental group, slices were obtained from four to five animals. The rate of DA clearance, reported as a rate constant k, was significantly and dose-dependently decreased by AMPH in both the core and shell of the NAc in WT mice. AMPH had no effect on DA clearance in either the NAc core or shell of DAT-KO mice. The data are presented as mean ± SEM. Differences with P < 0.05 are indicated with asterisks.

Fig. 4.

Mean firing rate and response to AMPH of dopaminergic VTA neurons from WT mice (control) and DAT-KO mice. The responses to AMPH of DA-VTA neurons from WT and DAT-KO mice were averaged and plotted as a function of AMPH concentration. The mean spontaneous firing rate of DA-VTA neurons from WT mice was reduced by AMPH in a concentration-dependent manner over the concentration range of 1-10 μM. In contrast, over the concentration range of AMPH that was tested (2-100 μM), no significant inhibition of firing was observed. Asterisks indicate a significant difference in the AMPH response of DA-VTA neurons from WT mice compared with those from DAT-KO mice. (Inset) The mean firing rate of DA-VTA neurons from WT and DAT-KO mice. Before the addition of AMPH, the spontaneous firing rate of DA-VTA neurons from DAT-KO mice was not significantly different from that of WT mice. The baseline firing rate of the control DA-VTA neurons was 1.9 ± 0.3 Hz (n = 10), and the baseline firing rate of the DA-VTA neurons from DAT-KO mice was 1.8 ± 0.3 Hz (n = 7).

Fig. 5.

Rewarding effect of AMPH in the DAT-KO and WT mice. (A) Place preference conditioning of DAT-KO and WT mice (n = 7) with saline or AMPH (5 mg/kg) administration over a 4-day period (four pairings). (B) Place preference conditioning of DAT-KO and WT mice (n = 7-8) with saline or AMPH (2.5 or 5 mg/kg) administration over a 2-day period (two pairings). Time scores shown represent differences between postconditioning and preconditioning time spent in the saline- or drug-paired environment. ^*, P < 0.05 between AMPH and saline. @, P = 0.08 between AMPH and saline.

Fig. 6.

WAY-100635 (WAY) abolishes AMPH-induced CPP in DAT-KO but not in WT mice. The left side of the graph shows that WT mice (n = 7-8) exhibited robust AMPH CPP (5 mg/kg, also shown in Fig. 5A), and WAY-100635 pretreatment did not affect place conditioning. The right side of the graph shows that DAT-KO mice (n = 7-8) also exhibited robust AMPH CPP (5 mg/kg), and WAY-100635 (2 mg/kg) abolished this effect. Time scores shown represent differences between postconditioning and preconditioning time spent in the saline- or drug-paired environment. ^*, significant difference from saline control (P < 0.05).