Abolished cocaine reward in mice with a cocaine-insensitive dopamine transporter

Abstract

There are three known high-affinity targets for cocaine: the dopamine transporter (DAT), the serotonin transporter (SERT), and the norepinephrine transporter (NET). Decades of studies support the dopamine (DA) hypothesis that the blockade of DAT and the subsequent increase in extracellular DA primarily mediate cocaine reward and reinforcement. Contrary to expectations, DAT knockout (DAT-KO) mice and SERT or NET knockout mice still self-administer cocaine and/or display conditioned place preference (CPP) to cocaine, which led to the reevaluation of the DA hypothesis and the proposal of redundant reward pathways. To study the role of DAT in cocaine reward, we have generated a knockin mouse line carrying a functional DAT that is insensitive to cocaine. In these mice, cocaine suppressed locomotor activity, did not elevate extracellular DA in the nucleus accumbens, and did not produce reward as measured by CPP. This result suggests that blockade of DAT is necessary for cocaine reward in mice with a functional DAT. This mouse model is unique in that it is specifically designed to differentiate the role of DAT from the roles of NET and SERT in cocaine-induced biochemical and behavioral effects.

Fig. 1.

Generation of DAT mutant knockin mice. (A) Targeting strategy. Thin lines, mouse genomic DNA; thick lines, sequences included in the targeting construct; K, KpnI sites; open box, mDAT exon 3; open box with a line, exon 3 with the triple mutation L104V/F105C/A109V and a KpnI site; open arrow, Neo cassette; triangles, LoxP sites; shaded arrow, thymidine kinase gene; shaded box, probe for Southern blot analysis; arrows, PCR primers. (B) PCR using primer f1 external of the short arm and mutant-specific primer r1. (C) PCR using specific primer f2 and primer r2 external of the long arm. (D) PCR using primers f3 and r3, which amplify a 564-bp fragment from the WT allele and a 667-bp fragment from the mutant allele. (E) Southern blot analysis of mouse genomic DNA digested with KpnI; the additional KpnI in the mutant allele reduces the probed fragment from 4.2 to 3.4 kb. HET, heterozygous; DAT-CI, homozygous mutant mice with cocaine-insensitive DAT; M, DNA marker.

Fig. 2.

Expression and pharmacological properties of mutant DAT in the knockin mice. (A and B) Northern (A) and Western (B) blot analysis of total (Left) or cell surface (Right) proteins did not reveal any significant difference (P > 0.05, t test; n = 3–5) in DAT expression levels between WT and DAT-CI mice. DAT activity was measured in striatal synaptosomes in the presence of increasing concentrations of cocaine or unlabeled DA. (C) The mutant DAT was 89-fold more insensitive to cocaine inhibition than the WT DAT. (D) The maximum DA uptake activities (V_max) did not differ significantly between DATs from WT and DAT-CI mice, but apparent affinity (K_m) of the mutant DAT was significantly higher (t test; n = 7) than that of the WT DAT.

Fig. 3.

Differential effects of cocaine on DA release, uptake, and DA neuron firing between genotypes measured in vitro and in vivo. (A) FCV was used to measure DA release and reuptake in accumbal slices in the absence or presence of 4 μM cocaine. Cocaine prolonged the DA decay time from brain slices of WT mice but not of DAT-CI mice. (B) The frequencies of spontaneous action potentials in DA neurons were recorded with whole-cell patch clamp and presented as mean ± SEM. The effects of bath application of 5 μM DA and 5 μM cocaine are shown. Cocaine significantly decreased the frequency of DA neuron firing in WT mice but not in DAT-CI mice. Insets show actual spike traces. Spikes were truncated for display. (Scale bars: 500 ms and 20 mV.) (C) Extracellular DA in the NAc was assessed by microdialysis in free-moving mice. The average of the two samples before the treatment (i.p.) of cocaine (20 mg/kg), AMPH (2.5 mg/kg), or saline was used as the baseline (100%, 0 min). Cocaine significantly increased extracellular DA level in the NAc of WT mice (F_1,113 = 28.4; P < 0.0001) but not in DAT-CI mice (F_1,113 = 2.89; P > 0.05). AMPH elevated extracellular DA level in both WT (F_1,81 = 52.0; P < 0.0001) and DAT-CI (F_1,81 = 6.5; P < 0.05) mice. Two-way ANOVAs were performed. Data represent mean ± SEM (n = 5–8).

Fig. 4.

Locomotor activity in WT and DAT-CI mice. The locomotor activities are presented as horizontal distances traveled (in centimeters) in 15 or 120 min (mean ± SEM) after drug injection in mice (n = 6–12). (A) Cocaine stimulated locomotion (15 min) in WT mice but suppressed it in DAT-CI mice (for genotype difference, F_1,33 = 32.16 and P < 0.0001; for genotype × drug interaction, F_2,33 = 21.84 and P < 0.0001). (B) AMPH elevated locomotor activity (15 min) in both WT and DAT-CI mice (for drug effect, F_2,45 = 29.20 and P < 0.0001). (C) Morphine also stimulated locomotion (120 min) in both genotypes (for drug effect, F_1,29 = 28.54 and P < 0.0001). ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001 between drug and saline in the same genotype.

Fig. 5.

Drug-induced CPP in WT and DAT-CI mice. CPP is represented by the time difference (in seconds) between pre- and postconditioning (mean ± SEM) that mice spend in the drug-paired chamber (n = 8–15). (A) Five and 20 mg/kg cocaine produced significant CPP in WT mice but not in DAT-CI mice compared with saline mice (for genotype difference, F_1,66 = 49.96 and P < 0.0001; for genotype × drug interaction, F_2,66 = 16.7 and P < 0.0001). (B) AMPH (2.5 mg/kg) produced CPP in both WT and DAT-CI mice (for drug effect, F_1,47 = 18.86 and P < 0.0001). ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001 between drug and saline in the same genotype; n = 8–15.